Nuclease-mediated regulation of gene expression

ABSTRACT

The present disclosure is in the field of genome engineering, particularly targeted modification of the genome of a hematopoietic cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/565,811, filed Oct. 11, 2017, which is a 35 U.S.C. § 371 filing ofPCT/US2016/032049, filed May 12, 2016, which claims the benefit of U.S.Provisional Application No. 62/160,396, filed May 12, 2015 and U.S.Provisional Application No. 62/303,595, filed Mar. 4, 2016, thedisclosures of which are hereby incorporated by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 15, 2020, isnamed 8328013201_SL.txt and is 9,688 bytes in size.

TECHNICAL FIELD

The present disclosure is in the field of genome engineering,particularly targeted modification of the genome of a hematopoieticcell.

BACKGROUND

When one considers that genome sequencing efforts have revealed that thehuman genome contains between 20,000 and 25,000 genes, but fewer than2000 transcriptional regulators, it becomes clear that a number offactors must interact to control gene expression in all its varioustemporal, developmental and tissue specific manifestations. Expressionof genes is controlled by a highly complex mixture of general andspecific transcriptional regulators and expression can also becontrolled by cis-acting DNA elements. These DNA elements comprise bothlocal DNA elements such as the core promoter and its associatedtranscription factor binding sites as well as distal elements such asenhancers, silencers, insulators and locus control regions (LCRs) (seeMatson et al. (2006) Ann Rev Genome Hum Genet 7:29-59).

Enhancer elements were first identified in the SV40 viral genome, andthen found in the human immunoglobulin heavy chain locus. Now known toplay regulatory roles in the expression of many genes, enhancers appearto mainly influence temporal and spatial patterns of gene expression. Ithas also been found that enhancers function in a manner that is notdependent upon distance from the core promoter of a gene, and is notdependent on any specific sequence orientation with respect to thepromoter. Enhancers can be located several hundred kilobases upstream ordownstream of a core promoter region, where they can be located in anintron sequence, or even beyond the 3′ end of a gene.

Various methods and compositions for targeted cleavage of genomic DNAhave been described. Such targeted cleavage events can be used, forexample, to induce targeted mutagenesis, induce targeted deletions ofcellular DNA sequences, and facilitate targeted recombination at apredetermined chromosomal locus. See, e.g., U.S. Pat. Nos. 9,255,250;9,200,266; 9,045,763; 9,005,973; 9,150,847; 8,956,828; 8,945,868;8,703,489; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558;7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925;8,110,379; 8,409,861; U.S. Patent Publication Nos. 2003/0232410;2005/0208489; 2005/0026157; 2005/0064474; 2006/0063231; 2008/0159996;2010/00218264; 2012/0017290; 2011/0265198; 2013/0137104; 2013/0122591;2013/0177983; 2013/0196373; 2015/0056705 and 2015/0335708, thedisclosures of which are incorporated by reference in their entireties.

These methods often involve the use of engineered cleavage systems toinduce a double strand break (DSB) or a nick in a target DNA sequencesuch that repair of the break by an error born process such asnon-homologous end joining (NHEJ) or repair using a repair template(homology directed repair or HDR) can result in the knock out of a geneor the insertion of a sequence of interest (targeted integration). Thistechnique can also be used to introduce site specific changes in thegenome sequence through use of a donor oligonucleotide, including theintroduction of specific deletions of genomic regions, or of specificpoint mutations or localized alterations (also known as genecorrection). Cleavage can occur through the use of specific nucleasessuch as engineered zinc finger nucleases (ZFN), transcription-activatorlike effector nucleases (TALENs), or using the CRISPR/Cas system with anengineered crRNA/tracr RNA (‘single guide RNA’) to guide specificcleavage. Further, targeted nucleases are being developed based on theArgonaute system (e.g., from T. thermophilus, known as ‘TtAgo’, seeSwarts et al. (2014) Nature 507(7491): 258-261), which also may have thepotential for uses in genome editing and gene therapy.

Red blood cells (RBCs), or erythrocytes, are the major cellularcomponent of blood. In fact, RBCs account for one quarter of the cellsin a human. Mature RBCs lack a nucleus and many other organelles inhumans, and are full of hemoglobin, a metalloprotein that functions tocarry oxygen to the tissues as well as carry carbon dioxide out of thetissues and back to the lungs for removal. This protein makes upapproximately 97% of the dry weight of RBCs and it increases the oxygencarrying ability of blood by about seventy fold. Hemoglobin is aheterotetramer comprising two alpha (α)-like globin chains and two beta(β)-like globin chains and 4 heme groups. In adults the α2β2 tetramer isreferred to as Hemoglobin A (HbA) or adult hemoglobin. Typically, thealpha and beta globin chains are synthesized in an approximate 1:1 ratioand this ratio seems to be critical in terms of hemoglobin and RBCstabilization. In a developing fetus, a different form of hemoglobin,fetal hemoglobin (HbF), is produced which has a higher binding affinityfor oxygen than Hemoglobin A such that oxygen can be delivered to thebaby's system via the mother's blood stream. There are two genes thatencode fetal globin that are very similar in sequence and are termedHPG1 (also referred to as Ggamma) and HPG2 (Agamma). Fetal hemoglobinprotein also contains two α globin chains, but in place of the adultβ-globin chains, it has two fetal gamma (γ)-globin chains (i.e., fetalhemoglobin is α2γ2). At approximately 30 weeks of gestation, thesynthesis of gamma globin in the fetus starts to drop while theproduction of beta globin increases. By approximately 10 months of age,the newborn's hemoglobin is nearly all α2β2 although some HbF persistsinto adulthood (approximately 1-3% of total hemoglobin). The regulationof the switch from production of gamma- to beta-globin is quite complex,and primarily involves a down-regulation of gamma globin transcriptionwith a simultaneous up-regulation of beta globin transcription.

Genetic defects in the sequences encoding the hemoglobin chains can beresponsible for a number of diseases known as hemoglobinopathies,including sickle cell anemia and thalassemias. In the majority ofpatients with hemoglobinopathies, the genes encoding gamma globin remainpresent, but expression is relatively low due to normal gene repressionoccurring around parturition as described above.

It is estimated that 1 in 5000 people in the U.S. have sickle celldisease (SCD), mostly in people of sub-Saharan Africa descent. Thereappears to be a benefit for heterozygous carriers of the sickle cellmutation for protection against malaria, so this trait may have beenpositively selected over time, such that it is estimated that insub-Saharan Africa, one third of the population has the sickle celltrait. Sickle cell disease is caused by a mutation in the 0 globin geneas a consequence of which valine is substituted for glutamic acid atamino acid #6 (a GAG to GTG at the DNA level), where the resultanthemoglobin is referred to as “hemoglobinS” or “HbS.” Under lower oxygenconditions, a conformational shift in the deoxy form of HbS exposes ahydrophobic patch on the protein between the E and F helices. Thehydrophobic residues of the valine at position 6 of the beta chain inhemoglobin are able to associate with the hydrophobic patch, causing HbSmolecules to aggregate and form fibrous precipitates. These aggregatesin turn cause the abnormality or ‘sickling’ of the RBCs, resulting in aloss of flexibility of the cells. The sickling RBCs are no longer ableto squeeze into the capillary beds and can result in vaso-occlusivecrisis in sickle cell patients. In addition, sickled RBCs are morefragile than normal RBCs, and tend towards hemolysis, eventually leadingto anemia in the patient.

Treatment and management of sickle cell patients is a life-longproposition involving antibiotic treatment, pain management andtransfusions during acute episodes. One approach is the use ofhydroxyurea, which exerts its effects in part by increasing theproduction of gamma globin. Long term side effects of chronichydroxyurea therapy are still unknown, however, and treatment givesunwanted side effects and can have variable efficacy from patient topatient. Despite an increase in the efficacy of sickle cell treatments,the life expectancy of patients is still only in the mid to late 50'sand the associated morbidities of the disease have a profound impact ona patient's quality of life.

Thalassemias are also diseases relating to hemoglobin and typicallyinvolve a reduced expression of globin chains. This can occur throughmutations in the regulatory regions of the genes or from a mutation in aglobin coding sequence that results in reduced expression or reducedlevels or functional globin protein. Alpha thalassemias are mainlyassociated with people of Western Africa and South Asian descent, andmay confer malarial resistance. Beta thalassemia is mainly associatedwith people of Mediterranean descent, typically from Greece and thecoastal areas of Turkey and Italy. In thalassemia minor, only one of theβ globin alleles bears a mutation. Individuals will suffer frommicrocytic anemia, and detection usually involves lower than normal meancorpuscular volume (<80 fL). The alleles of subjects with thalassemiaminor are β+/β or β0/β (where ‘β+’ refers to alleles that allow someamount of β chain formation to occur, ‘β’ refers to wild type β globinalleles, and ‘β0’ refers to β globin mutations comprising some form ofdeletion). Thalassemia intermedia subject can often manage a normal lifebut may need occasional transfusions, especially at times of illness orpregnancy, depending on the severity of their anemia. These patientsalleles can be β+/β+ or βo/β+. Thalassemia major occurs when bothalleles have thalassemia mutations. This is severely microcytic andhypochromic anemia. Untreated, it causes anemia, splenomegaly and severebone deformities. It progresses to death before age 20. Treatmentconsists of periodic blood transfusion; splenectomy for splenomegaly andchelation of transfusion-caused iron overload. Bone marrow transplantsare also being used for treatment of people with severe thalassemias ifan appropriate donor can be identified, but this procedure can havesignificant risks.

One approach that has been proposed for the treatment of both SCD andbeta thalassemias is to increase the expression of gamma globin with theaim to have HbF functionally replace the aberrant adult hemoglobin. Asmentioned above, treatment of SCD patients with hydroxyurea is thoughtto be successful in part due to its effect on increasing gamma globinexpression. The first group of compounds discovered to affect gammaglobin reactivation activity were cytotoxic drugs. The ability to causede novo synthesis of gamma-globin by pharmacological manipulation wasfirst shown using 5-azacytidine in experimental animals (DeSimone (1982)Proc Nat'l Acad Sci USA 79(14):4428-31). Subsequent studies confirmedthe ability of 5-azacytidine to increase HbF in patients withβ-thalassemia and sickle cell disease (Ley et al. (1982) N. Engl. J.Medicine 307:1469-1475 and Ley et al. (1983) Blood 62:370-380). Inaddition, short chain fatty acids (e.g. butyrate and derivatives) havebeen shown in experimental systems to increase HbF (Constantoulakis etal. (1988) Blood 72(6):1961-1967). Also, there is a segment of the humanpopulation with a condition known as ‘Hereditary Persistence of FetalHemoglobin’ (HPFH) where elevated amounts of HbF persist in adulthood(10-40% in HPFH heterozygotes (see Thein et al. (2009) Hum. Mol. Genet18(R2):R216-R223). This is a rare condition, but in the absence of anyassociated beta globin abnormalities, is not associated with anysignificant clinical manifestations, even when 100% of the individual'shemoglobin is HbF. When individuals that have a beta thalassemia alsohave co-incident HPFH, the expression of HbF can lessen the severity ofthe disease. Further, the severity of the natural course of sickle celldisease can vary significantly from patient to patient, and thisvariability, in part, can be traced to the fact that some individualswith milder disease express higher levels of HbF.

One approach to increase the expression of HbF involves identificationof genes whose products play a role in the regulation of gamma globinexpression. One such gene is BCL11A, first identified because of itsrole in lymphocyte development. BCL11A encodes a zinc finger proteinthat is thought to be involved in the developmental stage-specificregulation of gamma globin expression. BCL11A is expressed in adulterythroid precursor cells and down-regulation of its expression leads toan increase in gamma globin expression. In addition, it appears that thesplicing of the BCL11A mRNA is developmentally regulated. In embryoniccells, it appears that the shorter BCL11A mRNA variants, known asBCL11A-S and BCL11A-XS are primary expressed, while in adult cells, thelonger BCL11A-L and BCL11A-XL mRNA variants are predominantly expressed.See, Sankaran et al. (2008) Science 322:1839. The BCL11A protein appearsto interact with the beta globin locus to alter its conformation andthus its expression at different developmental stages. Use of aninhibitory RNA targeted to the BCL11A gene has been proposed (see, e.g.,U.S. Patent Publication No. 2011/0182867) but this technology hasseveral potential drawbacks, namely that complete knock down may not beachieved, delivery of such RNAs may be problematic and the RNAs must bepresent continuously, requiring multiple treatments for life.

Targeting of BCL11A enhancer sequences provides a mechanism forincreasing HbF. See, e.g., U.S. Patent Publication No. 2015/0132269.Genome wide association studies have identified a set of geneticvariations at BCL11A that are associated with increased HbF levels.These variations are a collection of SNPs found in non-coding regions ofBCL11A that function as a stage-specific, lineage-restricted enhancerregion. Further investigation revealed that this BCL11A enhancer isrequired in erythroid cells for BCL11A expression, but is not requiredfor its expression in B cells (see Bauer et al. (2013) Science342:253-257). The enhancer region was found within intron 2 of theBCL11A gene, and three areas of DNAsecI hypersensitivity (oftenindicative of a chromatin state that is associated with regulatorypotential) in intron 2 were identified. These three areas wereidentified as “+62”, “+58” and “+55” in accordance with the distance inkilobases from the transcription start site of BCL11A. These enhancerregions are roughly 350 (+55); 550 (+58); and 350 (+62) nucleotides inlength (Bauer 2013, ibid).

Thus, there remains a need for additional methods and compositions thatfor the alteration of BCL11A gene expression for example to treathemoglobinopathies such as sickle cell disease and beta thalassemia.

SUMMARY

The present invention describes compositions and methods for use in genetherapy and genome engineering. Specifically, the methods andcompositions described relate to inactivating (e.g., by completely orpartially abolishing its expression) a BCL11A gene, for example a genethat acts as regulator of one or more additional genes. In particular,the invention describes methods and compositions for interfering withenhancer function in a BCL11A gene to diminish or knock out its activityin specific cell lineages. Additionally, the invention provides methodsand compositions for interfering with BCL11A enhancer functions whereinthe enhancer sequences are not located within the BCL11A gene. Theresulting down-regulation of the BCL11A gene in these circumstances inturn results in increased expression of gamma globin.

In some aspects, the invention comprises a non-naturally occurring zincfinger protein comprising a zinc finger protein (ZFP) comprising 4, 5 or6 fingers, each finger comprising a recognition helix region thatrecognizes a target subsite wherein the recognition helix regionscomprise the sequences in the order shown in a single row of Table 1. Incertain embodiments, the ZFP comprises the recognition helixes as shownin Table 1 for the proteins designated as follows: 51446, 51463, 51484,51856, 51857 or 51862 (which bind to the target site shown in SEQ IDNO:1) and 51536, 51949, 51990, 51993, 51979, 51982, 52015, 52032 (whichbind to the target site shown in SEQ ID NO:12). Thus, in certainembodiments, provided herein is a zinc finger protein including thefollowing recognition helix regions:

(i) F1: (SEQ ID NO: 7) STGNLTN; F2: (SEQ ID NO: 5) TSGSLTR; F3:(SEQ ID NO: 2) DQSNLRA; and F4: (SEQ ID NO: 6) AQCCLFH; or (ii) F1:(SEQ ID NO: 2) DQSNLRA; F2: (SEQ ID NO: 3) RPYTLRL; F3: (SEQ ID NO: 8)SRGALKT; F4: (SEQ ID NO: 5) TSGSLTR; F5: (SEQ ID NO: 2) DQSNLRA; and F6:(SEQ ID NO: 6) AQCCLFH; (iii) F1: (SEQ ID NO: 2) DQSNLRA; F2:(SEQ ID NO: 9) RNFSLTM; F3: (SEQ ID NO: 10) SNGNLRN or (SEQ ID NO: 7)STGNLTN or (SEQ ID NO: 11) SSYNLAN; F4: (SEQ ID NO: 5) TSGSLTR; F5:(SEQ ID NO: 2) DQSNLRA; and F6: (SEQ ID NO: 6) AQCCLFH; or (iv) F1:(SEQ ID NO: 13) RSDHLTQ; F2: (SEQ ID NO: 14) QSGHLAR; F3:(SEQ ID NO: 15) QKGTLGE; F4: (SEQ ID NO: 18) RHRDLSR; and F5:(SEQ ID NO: 17) RRDNLHS; or (v) F1: (SEQ ID NO: 19) RNDHRTT; F2:(SEQ ID NO: 20) QKAHLIR; F3: (SEQ ID NO: 15) QKGTLGE; F4:(SEQ ID NO: 21) RGRDLSR or (SEQ ID NO: 25) LKRTLKR; and F5:(SEQ ID NO: 17) RRDNLHS; or (vi) F1: (SEQ ID NO: 13) RSDHLTQ; F2:(SEQ ID NO: 22) QRAHLTR; F3: (SEQ ID NO: 15) QKGTLGE or (SEQ ID NO: 24)QSGTRNH; F4: (SEQ ID NO: 23) HRNTLVR; and F5: (SEQ ID NO: 17) RRDNLHS;or (vii) F1: (SEQ ID NO: 13) RSDHLTQ; F2: (SEQ ID NO: 20) QKAHLIR; F3:(SEQ ID NO: 15) QKGTLGE or (SEQ ID NO: 24) QSGTRNH; F4: (SEQ ID NO: 21)RGRDLSR; and F5: (SEQ ID NO: 17) RRDNLHS; or (viii) F1: F1:(SEQ ID NO: 13) RSDHLTQ; F2: (SEQ ID NO: 14) QSGHLAR; F3:(SEQ ID NO: 24) QSGTRNH; F4: (SEQ ID NO: 16) QSSDLSR; and F5:(SEQ ID NO: 17) RRDNLHS.

In certain embodiments, the zinc finger proteins as described herein arefused to a functional domain (e.g., transcriptional activation domain,transcriptional repression domain, cleavage domain (to form a zincfinger nuclease), etc.). Zinc finger nucleases may be used in dimerizingpairs to cleave at or near one or both of the target sites for the ZFNsof the pair, for example “left partners” of Table 1 (e.g., 51446, 51463,51484, 51856, 51857, or 51862) can form dimers with the “right partners”of Table 1 (e.g., 51536, 51949, 51990, 51993, 51979, 51982, 52015, or52032) to cleave BCL11A enhancer sequences.

In another aspect, the invention comprises delivery of at least onenuclease (e.g., a nuclease that binds to a BCL11A enhancer sequence) toa human stem cell or precursor cell (HSC/PC) for the purpose of genomeengineering. In certain embodiments, the nuclease comprises a zincfinger protein (ZFP) comprising 4, 5 or 6 fingers, each fingercomprising a recognition helix region that recognizes a target subsitewherein the recognition helix regions comprise the sequences in theorder shown in a single row of Table 1. The nuclease(s) as describedherein may further comprise a linker (e.g., between the DNA-bindingdomain and the cleavage domain), for example a linker as shown in SEQ IDNOs:26-29 and U.S. Patent Publication No. 2015/0132269.

In some embodiments, the nuclease is delivered as a peptide, while inothers it is delivered as a nucleic acid encoding the at least onenuclease. In some embodiments, more than one nuclease is used. In somepreferred embodiments, the nucleic acid encoding the nuclease is anmRNA, and in some instances, the mRNA is protected. In some aspects, themRNA may be chemically modified (See e.g. Kormann et al. (2011) NatureBiotechnology 29(2):154-157). In other aspects, the mRNA may comprise anARCA cap (see U.S. Pat. Nos. 7,074,596 and 8,153,773). In furtherembodiments, the mRNA may comprise a mixture of unmodified and modifiednucleotides (see U.S. Patent Publication No. 2012/0195936). In apreferred embodiment, the nucleic acid encoding the nuclease(s) isdelivered to the HSC/PC via electroporation. In some embodiments, thenuclease cleaves at or near the binding site of transcription factor. Insome aspects, the transcription factor is GATA-1.

In other aspects, the invention comprises a cell or cell line in whichan endogenous BCL11A enhancer sequence is genetically modified by anuclease as described herein (e.g., shown in Table 1), for example ascompared to the wild-type sequence of the cell. Nuclease-modified cellsor cell lines as described herein are distinguishable in structureand/or function from both wild-type and other modified(nuclease-mediated) cells. The genetically modified cell or cell linesmay be heterozygous or homozygous for the modification. Themodifications may comprise insertions (e.g., transgene insertion),deletions and/or combinations thereof. In some preferred embodiments,the insertions, deletions and/or combinations thereof result in thedestruction of a transcription factor binding site. In certainembodiments, the modification is at or near the nuclease(s) bindingand/or cleavage site(s), for example, within 1-300 (or any valuetherebetween) base pairs upstream or downstream of the site(s) ofcleavage, more preferably within 1-100 base pairs (or any valuetherebetween) of either side of the binding and/or cleavage site(s)shown in Table 1, even more preferably within 1 to 50 base pairs (or anyvalue therebetween) on either side of the binding and/or cleavagesite(s). The modification may also include modifications to one or morenucleotides in the cleavage and/or in one or more of the binding sites.In certain embodiments, one or more of the nuclease target site(s)is(are) not modified. In other embodiments, at least one of the targetsites for the nuclease(s) is(are) modified. In certain embodiments, themodification is at or near the “+58” region of the BCL11A enhancer, forexample, at or near a nuclease binding site shown in any of SEQ ID NO:1and SEQ ID NO:12. Any cell or cell line may be modified by the nucleasesas described herein, for example a stem cell (hematopoietic stem cellsuch as a CD34+ hematopoietic stem cell) or red blood cell (RBC)precursor cell. Also described are cells or cell lines obtainedfollowing modification by a nuclease as described herein, for examplecells or cell lines descended from a nuclease-modified cell or cellline. Partially or fully differentiated cells descended from themodified stem cells as described herein are also provided (e.g., RBCs orRBC precursor cells). The cells descended from the nuclease-modifiedcells may be propagated (and/or differentiated) in vitro (culture) ormay differentiate within a live subject, for example following ex vivoadministration of a nuclease-modified stem cell. Any of the geneticallymodified cells or cell lines disclosed herein may show increasedexpression of gamma globin. Compositions such as pharmaceuticalcompositions comprising the genetically modified cells as describedherein are also provided.

In other aspects, the invention comprises delivery of a donor nucleicacid to a target cell to provide a genetically modified cell in whichthe donor is integrated into the cell. The donor may be delivered priorto, after, or along with the nucleic acid encoding the nuclease(s) ofTable 1. The donor nucleic acid may comprise an exogenous sequence(transgene) to be integrated into the genome of the cell, for example,an endogenous locus. In some embodiments, the donor may comprise a fulllength gene or fragment thereof flanked by regions of homology with thetargeted cleavage site. In some embodiments, the donor lacks homologousregions and is integrated into a target locus through homologyindependent mechanism (i.e. NHEJ). The donor may comprise any nucleicacid sequence, for example a nucleic acid that, when used as a substratefor homology-directed repair of the nuclease-induced double-strandbreak, leads to a donor-specified deletion to be generated at theendogenous chromosomal locus (e.g., BCL11A enhancer region) or,alternatively (or in addition to), novel allelic forms of (e.g., pointmutations that ablate a transcription factor binding site) theendogenous locus to be created. In some aspects, the donor nucleic acidis an oligonucleotide wherein integration leads to a gene correctionevent, or a targeted deletion.

In other aspects, the nuclease and/or donor is(are) delivered by viraland/or non-viral gene transfer methods. In preferred embodiments, thedonor is delivered to the cell via an adeno-associated virus (AAV). Insome instances, the AAV comprises LTRs that are of a heterologousserotype in comparison with the capsid serotype.

In some aspects, deletions comprising regions within the DNAseIhypersensitive regions of the enhancer (e.g., the +58 region of theBCL11A enhancer) are made using one or more nucleases as shown inTable 1. These deletions can comprise from about 1 nucleotide to about551 nucleotides. Thus, the deletions can comprise, 1, 5, 10, 15, 20, 25,30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550nucleotides, or any value therebetween. In some embodiments, thedeletions comprise binding regions for one or more transcriptionfactors. In some preferred embodiments, the deletions comprise a GATA-1binding site, or the binding site for GATA-1 in combination with otherfactors.

In some embodiments, the DNA binding domains of Table 1 are fused to afunctional domain. Some aspects include fusion of the DNA bindingdomains with domains capable of regulating the expression of a gene. Insome embodiments, the fusion proteins comprise the DNA binding domain ofTable 1 fused to a gene expression modulatory domain where the modulatorrepresses gene expression.

In some embodiments, the HSC/PC cells are contacted with the nucleasesand/or DNA binding proteins of the invention (i.e., ZFPs as shown inTable 1). In some embodiments, the nucleases and/or DNA binding proteinsare delivered as nucleic acids and in other embodiments, they aredelivered as proteins. In some embodiments, the nucleic acids are mRNAsencoding the nucleases and/or DNA binding proteins, and in furtherembodiments, the mRNAs may be protected. In some embodiments, the mRNAmay be chemically modified, may comprise an ARCA cap and/or may comprisea mixture of unmodified and modified nucleotides. Cells or cell linesdescended from these cells are also provided, including partially orfully differentiated cells.

In some aspects, the HSC/PC are contacted with the nucleases and/or DNAbinding proteins of the invention ex vivo, following apheresis of theHSC/PC from a subject, or purification from harvested bone marrow. Insome embodiments, the nucleases described herein cause modificationswithin the BCL11A enhancer regions, for example resulting a geneticallymodified cell that is structurally and/or functionally distinct fromwild-type and/or other modified (e.g., nuclease-modified) cells. Infurther embodiments, the HSC/PC containing the BCL11A enhancer regionmodifications are introduced back into the subject. In some instances,the HSC/PC containing the BCL11A enhancer region modifications areexpanded prior to introduction. In other aspects, the geneticallymodified HSC/PCs are given to the subject in a bone marrow transplantwherein the HSC/PC engraft, differentiate and mature in vivo. In someembodiments, the HSC/PC are isolated from the subject following G-CSF-and/or plerixafor-induced mobilization, and in others, the cells areisolated from human bone marrow or human umbilical cords. In someaspects, the subject is treated to a mild myeloablative procedure priorto introduction of the graft comprising the modified HSC/PC, while inother aspects, the subject is treated with a vigorous myeloablativeconditioning regimen. In some embodiments, the methods and compositionsof the invention are used to treat or prevent a hemoglobinopathy. Insome aspects, the hemoglobinopathy is a beta thalassemia, while in otheraspects, the hemoglobinopathy is sickle cell disease.

In some embodiments, the HSC/PC are further contacted with a donormolecule. In some embodiments, the donor molecule is delivered by aviral vector. The donor molecule may comprise one or more sequencesencoding a functional polypeptide (e.g., a cDNA or fragment thereof),with or without a promoter. Additional sequences (coding or non-codingsequences) may be included when a donor molecule is used forinactivation, including but not limited to, sequences encoding a 2Apeptide, SA site, IRES, etc.

In one aspect, the methods and compositions of the invention comprisemethods for contacting the HSC/PC in vivo. The nucleases and/or DNAbinding proteins are delivered to HSC/PC in situ by methods known in theart. In some embodiments, the nucleases and/or DNA binding proteins ofthe invention comprise a viral particle that is administered to thesubject in need, while in others, the nucleases and/or DNA bindingproteins comprise a nanoparticle (e.g. liposome). In some embodiments,the viral particles and/or nanoparticles are delivered to the organ(e.g. bone marrow) wherein the HSC/PC reside.

In another aspect, described herein are methods of integrating a donornucleic acid into the genome of a cell via homology-independentmechanisms. The methods comprise creating a double-stranded break (DSB)in the genome of a cell and cleaving the donor molecule using a nucleaseas described herein, such that the donor nucleic acid is integrated atthe site of the DSB. In certain embodiments, the donor nucleic acid isintegrated via non-homology dependent methods (e.g., NHEJ). As notedabove, upon in vivo cleavage the donor sequences can be integrated in atargeted manner into the genome of a cell at the location of a DSB. Thedonor sequence can include one or more of the same target sites for oneor more of the nucleases used to create the DSB. Thus, the donorsequence may be cleaved by one or more of the same nucleases used tocleave the endogenous gene into which integration is desired. In certainembodiments, the donor sequence includes different nuclease target sitesfrom the nucleases used to induce the DSB. DSBs in the genome of thetarget cell may be created by any mechanism. In certain embodiments, theDSB is created by one or more zinc-finger nucleases (ZFNs), fusionproteins comprising a zinc finger binding domain, which is engineered tobind a sequence within the region of interest, and a cleavage domain ora cleavage half-domain.

In one aspect, the donor may encode a regulatory protein of interest(e.g. ZFP TFs, TALE TFs or a CRISPR/Cas TF) that binds to and/ormodulates expression of a gene of interest. In one embodiment, theregulatory proteins bind to a DNA sequence and prevent binding of otherregulatory factors. In another embodiment, the binding of the regulatoryprotein may modulate (i.e. induce or repress) expression of a targetDNA.

In some embodiments, the transgenic HSC/PC cell and/or animal includes atransgene that encodes a human gene. In some instances, the transgenicanimal comprises a knock out at the endogenous locus corresponding toexogenous transgene, thereby allowing the development of an in vivosystem where the human protein may be studied in isolation. Suchtransgenic models may be used for screening purposes to identify smallmolecules or large biomolecules or other entities which may interactwith or modify the human protein of interest. In some aspects, thetransgene is integrated into the selected locus (e.g., safe-harbor) intoa stem cell (e.g., an embryonic stem cell, an induced pluripotent stemcell, a hematopoietic stem cell, etc.) or animal embryo obtained by anyof the methods described herein, and then the embryo is implanted suchthat a live animal is born. The animal is then raised to sexual maturityand allowed to produce offspring wherein at least some of the offspringcomprise edited endogenous gene sequence or the integrated transgene.

In another aspect, provided herein is a method of altering geneexpression (e.g., BCL11A and/or a globin gene) in a cell, the methodcomprising: introducing, into the cell, one or more nucleases asdescribed herein (shown in Table 1), under conditions such that the oneor more proteins are expressed and expression of the gene is altered. Incertain embodiments, expression of a globin gene (e.g., gamma globin orbeta globin) is altered (e.g., increased). Any of the methods describedherein may further comprise integrating a donor sequence (e.g.,transgene or fragment thereof under the control of an exogenous orendogenous promoter) into the genome of the cell, for exampleintegrating a donor at or near the site of nuclease cleavage in theBCL11A gene. The donor sequence is introduced to the cell using a viralvector, as an oligonucleotide and/or on a plasmid. The cell in whichgene expression is altered may be, for example, a red blood cell (RBC)precursor cell and/or a hematopoietic stem cell (e.g., CD34+ cell).

In other embodiments, provided herein is a method of producing agenetically modified cell comprising a genomic modification within anendogenous BCL11A enhancer sequence (a modification to the nucleotidesequence of the BCL11A enhancer sequence), the method comprising thesteps of: a) contacting a cell with a polynucleotide (e.g. DNA or mRNA)encoding a zinc finger nuclease comprising 4, 5, or 6 zinc fingerdomains in which each of the zinc finger domains comprises a recognitionhelix region in the order shown in a single row of Table 1; b)subjecting the cell to conditions conducive to expressing the zincfinger protein from the polynucleotide; and c) modifying the endogenousBCL11A enhancer sequence with the expressed zinc finger proteinsufficient to produce the genetically modified cell. In certainembodiments, the cells are stimulated with at least one cytokine (e.g.,prior to step (a)). The polynucleotide may be contacted with the cellusing any suitable method, including but not limited, via transfection,using a non-viral vector, using a viral vector, by chemical means or byexposure to an electric field (e.g., electroporation).

Cells comprising one or a combination of the genomic modificationsdescribed herein are also provided, including cells descended from thecells produced by the methods described herein.

Also provided is a method of treating a patient in need of an increasein globin gene expression, the method comprising administering to thepatient the pharmaceutical preparation (genetically modified cells,proteins and/or polynucleotides) as described herein in an amountsufficient to increase the globin gene expression in the patient. Incertain embodiments, the patient is known to have, is suspected ofhaving, or is at risk of developing a thalassemia or sickle celldisease.

A kit, comprising the nucleic acids, proteins and/or geneticallymodified cells of the invention, is also provided. The kit may comprisenucleic acids encoding the nucleases, (e.g. RNA molecules or ZFN, TALENor CRISPR/Cas system encoding genes contained in a suitable expressionvector), or aliquots of the nuclease proteins, donor molecules, suitablesternness modifiers, cells, buffers, and/or instructions (e.g., forperforming the methods of the invention) and the like. The inventionincludes, but is not limited to, a genetically modified cell (e.g., stemcell such as a hematopoietic (CD34+) stem cell or RBC precursor cell)comprising at least one genomic modification made by a nuclease (e.g.,as shown in a single row of Table 1), wherein the genomic modificationis within an endogenous BCL11A enhancer sequence, and further whereinthe genomic modification is selected from the group consisting ofinsertions, deletions and combinations thereof and comprises amodification at or near any of SEQ ID NO:1 and SEQ ID NO:12. In certainembodiments, the cell is a genetically modified differentiated celldescended from a stem cell as described herein (e.g., a RBC descendedfrom a hematopoietic stem cell or RBC precursor cell).

The nuclease may comprise at least one zinc finger nuclease (ZFN) (e.g.,as shown in Table 1) and/or at least one TALEN and the nuclease(s) maybe introduced into the cell in protein form and/or as a polynucleotideencoding the nuclease(s). In certain embodiments, the genomicmodification comprises an insertion that comprises integration of adonor polynucleotide encoding a transgene. Also provided arepharmaceutical compositions comprising one or more of the geneticallymodified cells as described herein.

Also provided is a DNA-binding protein comprising a zinc finger proteincomprising 4, 5 or 6 zinc finger domains comprising a recognition helixregion, wherein the zinc finger proteins comprise the recognition helixregions in the order shown in a single row of Table 1. Also provided isa TALE protein comprising a plurality of repeats that bind to a sequencecomprising a portion (e.g., at least 4, 5, 6 or more) base pairs of thetarget sites shown in Table 1. A fusion protein comprising a zinc fingerprotein or TALE protein as described herein and a wild-type orengineered cleavage domain or cleavage half-domain is also provided asare polynucleotides encoding the proteins (ZFPs, TALEs, ZFNs, TALENs) asdescribed herein. Cells (e.g., isolated stem cells such as hematopoietic(CD34+) stem cells) comprising one or more polynucleotides and/orproteins as described herein are also provided. Also provided are kitscomprising one or more proteins, polynucleotides and/or cells asdescribed herein.

A method of altering globin gene expression in a cell (e.g., RBCprecursor cell and/or hematopoietic stem cell) is also described, themethod comprising: introducing, into the cell, one or morepolynucleotides encoding one or more nucleases as described herein,under conditions such that the one or more proteins are expressed andexpression of the globin gene (e.g., gamma and/or beta globin) isaltered (e.g., increased). In certain embodiments, the methods furthercomprise integrating a donor sequence into the genome of the cell, forexample using a viral vector, as an oligonucleotide or on a plasmid. Thedonor sequence may comprise a transgene under the control of anendogenous or exogenous promoter.

Also provided is a method of producing a genetically modified cellcomprising a genomic modification within an endogenous BCL11A enhancersequence (e.g., target site as shown in Table 1), the method comprisingthe steps of: (a) contacting a cell with a polynucleotide encoding afusion protein comprising a zinc finger nuclease comprising 4, 5, or 6zinc finger domains in which each of the zinc finger domains comprises arecognition helix region in the order shown in a single row of Table 1;(b) subjecting the cell to conditions conducive to expressing the fusionprotein from the polynucleotide; and (c) modifying the endogenous BCL11Aenhancer sequence with the expressed fusion protein sufficient toproduce the genetically modified cell. In certain embodiments, themethod further comprises stimulating the cells with at least onecytokine. The polynucleotide(s) may be delivered inside the cell, forexample using a non-viral delivery system, a viral delivery system,and/or a delivery vehicle and may comprise subjecting the cells to anelectric field.

Methods of treating a patient in need of an increase in globin geneexpression (e.g., a patient is known to have, is suspected of having, oris at risk of developing a globinopathy such as a thalassemia (e.g.,β-thalassemia) or sickle cell disease are also provided, the methodcomprising administering to the patient the pharmaceutical compositionas described herein (e.g., proteins, polynucleotides and/or cells) in anamount sufficient to increase the globin gene expression in the patient.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the relative ratio of human gamma globinexpression (HBG) to human beta globin expression (HBB) in red bloodcells derived from CD34+ cells edited with the BCL11a-specific ZFN pairsshown.

FIGS. 2A and 2B are graphs depicting the activity of two pairs of BCL11aspecific ZFNs in CD34+ cells isolated from peripheral blood (PB). Cellswere transfected using a BTX electroporation device. The % indelsdetected (measurement of detectable NHEJ activity) for each conditionare shown below the graphs for FIG. 2A (mRNA input range from 0.5 to 4μg) and FIG. 2B (mRNA input range from 2.0 to 8.0 sg).

FIG. 3 is a graph depicting the expression of human gamma globin (HBG)as a relative ratio of HBG to human beta globin (HBB) followingerythroid differentiation of the edited PB CD34+ cells shown in FIG. 2B.Single mRNA species, where the ZFNs are encoded on the same mRNAmolecule but separated by a 2a self-cleaving peptide sequence(identified as “2a”), are compared to the use of two mRNAs where eachmRNA encodes one of the ZFN pair (identified as “sep” for separate).

FIG. 4A and FIG. 4B are graphs depicting the activity of two pairs ofBCL11a specific ZFNs in CD34+ cells isolated from bone marrow (BM).Cells were transfected using a BTX electroporation device. The % indelsdetected (measurement of detectable NHEJ activity) for each conditionare shown below the graphs for FIG. 4A (mRNA input range from 2.0 to 8.0μg). FIG. 4B depicts the activity of one pair of ZFNs where the ZFNs aresupplied either as a single mRNA species with a 2a self-cleaving peptidesequence separating the sequences encoding each ZFN or when the two ZFNsare supplied on separate mRNAs.

FIGS. 5A and 5B depicts activity of ZFN pairs in PB derived CD34+ cellsusing a Maxcyte electroporation device. The % indels detected(measurement of detectable NHEJ activity) for each condition are shownbelow the graphs for FIG. 5A and FIG. 5B. FIG. 5A depicts a comparisonbetween two ZFN pairs, and FIG. 5B depicts the activity of the ZFNspairs when the ZFNs are supplied either as a single mRNA species with a2a self-cleaving peptide sequence separating the sequences encoding eachZFN or when the two ZFNs are supplied on separate mRNAs.

FIG. 6 depicts a graph showing large scale activity of the A pair(SBS51446/51536) and B pair (SBS51857/51949) in bone marrow derivedCD34+ cells. mRNAs encoding the ZFN pairs were either supplied as singlemRNAs where the sequences encoding each half of the ZFN pair wereseparated by a 2a self-cleaving sequence, or as separate mRNAs encodingeach ZFN. Activity is shown in the % indels detected.

FIG. 7 shows a graph depicting the relative amount of HBG and HBBexpression detected after 14 days of differentiation following the largescale gene editing shown in FIG. 6. As before, samples were testedeither as single mRNAs encoding both ZFNs, or as separate mRNAs. Theamount of indels detected at day 0 of differentiation is shown acrossthe bottom, and demonstrates that indel activity tracks with the amountof HGB expressed.

FIG. 8 is a graph depicting the percent of indels detected in largescale editing of CD34+ cells from bone marrow treated with pair B,either as single mRNAs or separate mRNAs as described above.

FIGS. 9A and 9B are graphs depicting the percent of indels detected inlarge scale editing of CD34+ cells from bone marrow treated with pair B,either as single mRNAs or separate mRNAs as described above.

FIGS. 10A and 10B are tables depicting the results of the off-targetanalysis for Pair A (FIG. 10A) and Pair B (FIG. 10B).

FIGS. 11A and 11B are graphs showing real-time RT qPCR analysis of invitro differentiation in experiment 1 (FIG. 11A) and experiment 2 (FIG.11B) using patient and wild type (wt) cells treated with SB ZFN mRNA.The graphs show the relative ratios of gamma globin to alpha globinmRNAs.

FIGS. 12A and 12B are graphs showing the ratios of gamma globin to alphaglobin in experiment 1 (FIG. 12A) and experiment 2 (FIG. 12B). For thegamma globin values, the values of the Agamma and Ggamma peaks and,where applicable, the Agamma T peak were added up.

FIG. 13 shows a graph of the gamma/beta like protein ratios graphedaccording to the allele state in the individual colonies analyzed. Thedata were sorted by genotypic class (“+” for unmodified allele, “−” foredited allele; “+/+” for wild type; “+/−” for monoallelic modified; and“−/−” for biallelic modified).

DETAILED DESCRIPTION

Disclosed herein are compositions and methods for genome engineering forthe modulation of BCL11A and/or gamma globin expression and for thetreatment and/or prevention of hemoglobinopathies. In particular,nucleases comprising the ZFPs having the recognition helix regions asshown in a single row of Table 1 is efficiently achieved in HSC/PC andresults in a change in relative gamma globin expression duringsubsequent erythropoiesis. This modulation of BCL11A and gamma globinexpression is particularly useful for treatment of hemoglobinopathies(e.g., beta thalassemias, sickle cell disease) wherein there isinsufficient beta globin expression or expression of a mutated form ofbeta-globin. Using the methods and compositions of the invention, thecomplications and disease related sequelae caused by the aberrant betaglobin can be overcome by alteration of the expression of gamma globinin erythrocyte precursor cells.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind to anothermolecule. A binding protein can bind to, for example, a DNA molecule (aDNA-binding protein), an RNA molecule (an RNA-binding protein) and/or aprotein molecule (a protein-binding protein). In the case of aprotein-binding protein, it can bind to itself (to form homodimers,homotrimers, etc.) and/or it can bind to one or more molecules of adifferent protein or proteins. A binding protein can have more than onetype of binding activity. For example, zinc finger proteins haveDNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein.

Zinc finger and TALE binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger or TALE protein. Therefore, engineered DNA bindingproteins (zinc fingers or TALEs) are proteins that are non-naturallyoccurring. Non-limiting examples of methods for engineering DNA-bindingproteins are design and selection. A designed DNA binding protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPand/or TALE designs and binding data. See, for example, U.S. Pat. Nos.6,140,081; 6,453,242; 6,534,261 and 8,586,526; see also InternationalPatent Publication Nos. WO 98/53058; WO 98/53059; WO 98/53060; WO02/016536 and WO 03/016496.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759 and8,586,526; and International Patent Publication Nos. WO 95/19431; WO96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO01/88197 and WO 02/099084.

“TtAgo” is a prokaryotic Argonaute protein thought to be involved ingene silencing. TtAgo is derived from the bacteria Thermus thermophilus.See, e.g., Swarts et al., ibid, G. Sheng et al. (2013) Proc. Natl. Acad.Sci. U.S.A. 111:652). A “TtAgo system” is all the components requiredincluding, for example, guide DNAs for cleavage by a TtAgo enzyme.

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides, including but not limited to, donor captureby non-homologous end joining (NHFJ) and homologous recombination. Forthe purposes of this disclosure, “homologous recombination (HR)” refersto the specialized form of such exchange that takes place, for example,during repair of double-strand breaks in cells via homology-directedrepair mechanisms. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the one that experienced the double-strand break), and is variouslyknown as “non-crossover gene conversion” or “short tract geneconversion,” because it leads to the transfer of genetic informationfrom the donor to the target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or “synthesis-dependent strand annealing,” in which the donor isused to resynthesize genetic information that will become part of thetarget, and/or related processes. Such specialized HR often results inan alteration of the sequence of the target molecule such that part orall of the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide.

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a double-stranded break (DSB) in the targetsequence (e.g., cellular chromatin) at a predetermined site. The DSB mayresult in deletions and/or insertions by homology-directed repair or bynon-homology-directed repair mechanisms. Deletions may include anynumber of base pairs. Similarly, insertions may include any number ofbase pairs including, for example, integration of a “donor”polynucleotide, optionally having homology to the nucleotide sequence inthe region of the break. The donor sequence may be physically integratedor, alternatively, the donor polynucleotide is used as a template forrepair of the break via homologous recombination, resulting in theintroduction of all or part of the nucleotide sequence as in the donorinto the cellular chromatin. Thus, a first sequence in cellularchromatin can be altered and, in certain embodiments, can be convertedinto a sequence present in a donor polynucleotide. Thus, the use of theterms “replace” or “replacement” can be understood to representreplacement of one nucleotide sequence by another, (i.e., replacement ofa sequence in the informational sense), and does not necessarily requirephysical or chemical replacement of one polynucleotide by another.

In any of the methods described herein, additional pairs of zinc-fingerproteins or TALEN can be used for additional double-stranded cleavage ofadditional target sites within the cell.

Any of the methods described herein can be used for insertion of a donorof any size and/or partial or complete inactivation of one or moretarget sequences in a cell by targeted integration of donor sequencethat disrupts expression of the gene(s) of interest. Cell lines withpartially or completely inactivated genes are also provided.

In any of the methods described herein, the exogenous nucleotidesequence (the “donor sequence” or “transgene”) can contain sequencesthat are homologous, but not identical, to genomic sequences in theregion of interest, thereby stimulating homologous recombination toinsert a non-identical sequence in the region of interest. Thus, incertain embodiments, portions of the donor sequence that are homologousto sequences in the region of interest exhibit between about 80 to 99%(or any integer therebetween) sequence identity to the genomic sequencethat is replaced. In other embodiments, the homology between the donorand genomic sequence is higher than 99%, for example if only 1nucleotide differs as between donor and genomic sequences of over 100contiguous base pairs. In certain cases, a non-homologous portion of thedonor sequence can contain sequences not present in the region ofinterest, such that new sequences are introduced into the region ofinterest. In these instances, the non-homologous sequence is generallyflanked by sequences of 50-1,000 base pairs (or any integral valuetherebetween) or any number of base pairs greater than 1,000, that arehomologous or identical to sequences in the region of interest. In otherembodiments, the donor sequence is non-homologous to the first sequence,and is inserted into the genome by non-homologous recombinationmechanisms.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474, 2007/0218528, 2008/0131962and 2011/0201055, incorporated herein by reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 100,000,000 nucleotidesin length (or any integer value therebetween or thereabove), preferablybetween about 100 and 100,000 nucleotides in length (or any integertherebetween), more preferably between about 2000 and 20,000 nucleotidesin length (or any value therebetween) and even more preferable, betweenabout 5 and 15 kb (or any value therebetween).

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome” is a chromatin complex comprising all or a portion of thegenome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

An “accessible region” is a site in cellular chromatin in which a targetsite present in the nucleic acid can be bound by an exogenous moleculewhich recognizes the target site. Without wishing to be bound by anyparticular theory, it is believed that an accessible region is one thatis not packaged into a nucleosomal structure. The distinct structure ofan accessible region can often be detected by its sensitivity tochemical and enzymatic probes, for example, nucleases.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular, and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogenous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequence may beintroduced into a cell line originally derived from a mouse or hamster.Methods for the introduction of exogenous molecules into plant cells areknown to those of skill in the art and include, but are not limited to,protoplast transformation, silicon carbide (e.g., WHISKERS™),Agrobacterium-mediated transformation, lipid-mediated transfer (i.e.,liposomes, including neutral and cationic lipids), electroporation,direct injection, cell fusion, particle bombardment (e.g., using a “genegun”), calcium phosphate co-precipitation, DEAE-dextran-mediatedtransfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

As used herein, the term “product of an exogenous nucleic acid” includesboth polynucleotide and polypeptide products, for example, transcriptionproducts (polynucleotides such as RNA) and translation products(polypeptides).

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPor TALE DNA-binding domain and one or more activation domains) andfusion nucleic acids (for example, a nucleic acid encoding the fusionprotein described supra). Examples of the second type of fusion moleculeinclude, but are not limited to, a fusion between a triplex-formingnucleic acid and a polypeptide, and a fusion between a minor groovebinder and a nucleic acid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, random mutation) can be used to modulateexpression. Gene inactivation refers to any reduction in gene expressionas compared to a cell that does not include a ZFP, TALE or CRISPR/Cassystem as described herein. Thus, gene inactivation may be partial orcomplete.

A “protected” mRNA is one in which the mRNA has been altered in somemanner to increase the stability or translation of the mRNA. Examples ofprotections include the use of replacement of up to 25% of the cytodineand uridine residues with 2-thiouridine (s2U) and 5-methylcytidine(m5C). The resulting mRNA exhibits less immunogenicity and morestability as compared with its unmodified counterpart. (see Karikó etal. (2012) Molecular Therapy 16(11):1833-1844). Other changes includethe addition of a so-called ARCA cap, which increases thetranslationability of the in vitro produced mRNA (see U.S. Pat. No.7,074,596).

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFP, TALEor Cas DNA-binding domain is fused to an activation domain, the ZFP,TALE or Cas DNA-binding domain and the activation domain are inoperative linkage if, in the fusion polypeptide, the ZFP, TALE of CasDNA-binding domain portion is able to bind its target site and/or itsbinding site, while the activation domain is able to upregulate geneexpression. When a fusion polypeptide in which a ZFP, TALE or CasDNA-binding domain is fused to a cleavage domain, the ZFP, TALE or CasDNA-binding domain and the cleavage domain are in operative linkage if,in the fusion polypeptide, the ZFP, TALE or Cas DNA-binding domainportion is able to bind its target site and/or its binding site, whilethe cleavage domain is able to cleave DNA in the vicinity of the targetsite.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

The terms “subject” and “patient” are used interchangeably and refer tomammals such as human patients and non-human primates, as well asexperimental animals such as rabbits, dogs, cats, rats, mice, and otheranimals. Accordingly, the term “subject” or “patient” as used hereinmeans any mammalian patient or subject to which the or stem cells of theinvention can be administered. Subjects of the present invention includethose that have been exposed to one or more chemical toxins, including,for example, a nerve toxin.

“Sternness” refers to the relative ability of any cell to act in a stemcell-like manner, i.e., the degree of toti-, pluri-, or oligo-potencyand expanded or indefinite self-renewal that any particular stem cellmay have.

Nucleases

Described herein are compositions, particularly nucleases, that areuseful for in vivo cleavage of a donor molecule carrying a transgene andnucleases for cleavage of the genome of a cell such that the transgeneis integrated into the genome in a targeted manner. In certainembodiments, one or more of the nucleases are naturally occurring. Inother embodiments, one or more of the nucleases are non-naturallyoccurring, i.e., engineered in the DNA-binding domain and/or cleavagedomain. For example, the DNA-binding domain of a naturally-occurringnuclease may be altered to bind to a selected target site (e.g., ameganuclease that has been engineered to bind to site different than thecognate binding site). In other embodiments, the nuclease comprisesheterologous DNA-binding and cleavage domains (e.g., zinc fingernucleases; TAL-effector domain DNA binding proteins; meganucleaseDNA-binding domains with heterologous cleavage domains).

A. DNA-Binding Domains

In certain embodiments, the DNA binding domain of one or more of thenucleases used for in vivo cleavage and/or targeted cleavage of thegenome of a cell comprises a zinc finger protein. Preferably, the zincfinger protein is non-naturally occurring in that it is engineered tobind to a target site of choice. See, for example, See, for example,Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001)Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol.19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Chooet al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos.6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215;6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635 and 7,253,273; andU.S. Patent Publication Nos. 2005/0064474; 2007/0218528 and2005/0267061, all incorporated herein by reference in their entireties.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759 and 6,242,568; aswell as International Patent Publication Nos. WO 98/37186; WO 98/53057;WO 00/27878 and WO 01/88197 and GB Patent No. 2,338,237. In addition,enhancement of binding specificity for zinc finger binding domains hasbeen described, for example, in co-owned International PatentPublication No. WO 02/077227.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185 and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. Pat. Nos.6,140,081; 5,789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988;6,013,453 and 6,200,759; International Patent Publication Nos. WO95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO01/60970; WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO98/53060; WO 02/016536 and WO 03/016496.

Nearly any linker (spacer) may be used between one or more of thecomponents of the DNA-binding domain (e.g., zinc fingers), between oneor more DNA-binding domains and/or between the DNA-binding domain andthe functional domain (e.g., nuclease). Non-limiting examples ofsuitable linker sequences include U.S. Pat. Nos. 8,772,453; 7,888,121;6,479,626; 6,903,185; and 7,153,949; and U.S. Patent Publication Nos.2009/0305419; 2015/0064789 and 2015/0132269. Thus, the proteinsdescribed herein may include any combination of suitable linkers betweenthe individual DNA-binding components and/or between the DNA-bindingdomain and the functional domain of the compositions described herein.

B. Cleavage Domains

Any suitable cleavage domain can be operatively linked to theDNA-binding domains as described herein to form a nuclease. The cleavagedomain may be heterologous to the DNA-binding domain, for example a zincfinger DNA-binding domain and a cleavage domain from a nuclease.Heterologous cleavage domains can be obtained from any endonuclease orexonuclease. Exemplary endonucleases from which a cleavage domain can bederived include, but are not limited to, restriction endonucleases andhoming endonucleases. See, for example, 2002-2003 Catalogue, New EnglandBiolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res.25:3379-3388. Additional enzymes which cleave DNA are known (e.g., S1Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease;yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, ColdSpring Harbor Laboratory Press, 1993). One or more of these enzymes (orfunctional fragments thereof) can be used as a source of cleavagedomains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IS enzyme FokI catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as wellas Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is FokI. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95:10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the FokI enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-FokI fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and twoFokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-FokI fusionsare provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in U.S. Pat. No.7,888,121 incorporated herein in its entirety. Additional restrictionenzymes also contain separable binding and cleavage domains, and theseare contemplated by the present disclosure. See, for example, Roberts etal. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in See, e.g., U.S. Pat. Nos. 7,914,796; 8,034,598 and8,623,618, the disclosures of all of which are incorporated by referencein their entireties herein. Amino acid residues at positions 446, 447,479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537,and 538 of FokI are all targets for influencing dimerization of the FokIcleavage half-domains.

Exemplary engineered cleavage half-domains of FokI that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFokI and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (1) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Patent Publication No. 2008/0131962, the disclosure of which isincorporated by reference in its entirety for all purposes. In certainembodiments, the engineered cleavage half-domain comprises mutations atpositions 486, 499 and 496 (numbered relative to wild-type FokI), forinstance mutations that replace the wild type Gln (Q) residue atposition 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (1) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively. See, e.g., U.S. Pat. Nos. 7,914,796; 8,034,598and 8,623,618. In other embodiments, the engineered cleavage half domaincomprises the “Sharkey” and/or “Sharkey mutations” (see Guo et al.(2010) J. Mol. Biol. 400(l):96-107).

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (FokI) as described in U.S. Pat. Nos.7,888,121; 7,914,796; 8,034,598 and 8,623,618.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see, e.g. U.S.Patent Publication No. 2009/0068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in International PatentPublication No. WO 2009/042163 and U.S. Patent Publication No.2009/0068164. Expression of the nuclease may be under the control of aconstitutive promoter or an inducible promoter, for example thegalactokinase promoter which is activated (de-repressed) in the presenceof raffinose and/or galactose and repressed in presence of glucose.

The nuclease(s) as described herein may make one or more double-strandedand/or single-stranded cuts in the target site. In certain embodiments,the nuclease comprises a catalytically inactive cleavage domain (e.g.,FokI and/or Cas protein). See, e.g., U.S. Pat. Nos. 9,200,266 and8,703,489 and Guillinger et al. (2014) Nature Biotech. 32(6):577-582.The catalytically inactive cleavage domain may, in combination with acatalytically active domain act as a nickase to make a single-strandedcut. Therefore, two nickases can be used in combination to make adouble-stranded cut in a specific region. Additional nickases are alsoknown in the art, for example, McCaffery et al. (2016) Nucleic AcidsRes. 44(2):e11. doi: 10.1093/nar/gkv878. Epub 2015 Oct. 19.

Target Sites

As described in detail above, DNA domains can be engineered to bind toany sequence of choice. An engineered DNA-binding domain can have anovel binding specificity, compared to a naturally-occurring DNA-bindingdomain. In certain embodiments, the DNA-binding domains bind to asequence within a BCL11A enhancer sequence, for example a target site(typically 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or evenmore base pairs) is between exon 2 and exon 3 of BCL11A, includingDNA-binding domains that bind to a sequence within a DNAseIhypersensitive site in the BCL11A enhancer sequence (e.g., +58) as shownin Table 1. Engineering methods include, but are not limited to,rational design and various types of selection. Rational designincludes, for example, using databases comprising triplet (orquadruplet) nucleotide sequences and individual zinc finger amino acidsequences, in which each triplet or quadruplet nucleotide sequence isassociated with one or more amino acid sequences of zinc fingers whichbind the particular triplet or quadruplet sequence. See, for example,co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated byreference herein in their entireties. Rational design of TAL-effectordomains can also be performed. See, e.g., U.S. Patent Publication No.2011/0301073.

Exemplary selection methods applicable to DNA-binding domains, includingphage display and two-hybrid systems, are disclosed in U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466;6,200,759 and 6,242,568; as well as International Patent PublicationNos. WO 98/37186; WO 98/53057; WO 00/27878 and WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-ownedInternational Patent Publication No. WO 02/077227.

Selection of target sites; nucleases and methods for design andconstruction of fusion proteins (and polynucleotides encoding same) areknown to those of skill in the art and described in detail in U.S.Patent Publication Nos. 2005/0064474 and 2006/0188987, incorporated byreference in their entireties herein.

In addition, as disclosed in these and other references, DNA-bindingdomains (e.g., multi-fingered zinc finger proteins) and/or fusions ofDNA-binding domain(s) and functional domain(s) may be linked togetherusing any suitable linker sequences, including for example, linkers of 5or more amino acids. U.S. Pat. Nos. 8,772,453; 7,888,121 (e.g., “ZC”linker); U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949; U.S. PatentPublication Nos. 2009/0305419) and 2015/0064789. The proteins describedherein may include any combination of suitable linkers between theindividual DNA-binding domains of the protein. See, also, U.S. Pat. No.8,586,526.

Donors

In certain embodiments, the present disclosure relates tonuclease-mediated targeted integration of an exogenous sequence into thegenome of a cell using the BCL11A enhancer region-binding moleculesdescribed herein. As noted above, insertion of an exogenous sequence(also called a “donor sequence” or “donor” or “transgene”), for examplefor deletion of a specified region and/or correction of a mutant gene orfor increased expression of a wild-type gene. It will be readilyapparent that the donor sequence is typically not identical to thegenomic sequence where it is placed. A donor sequence can contain anon-homologous sequence flanked by two regions of homology to allow forefficient HDR at the location of interest or can be integrated vianon-homology directed repair mechanisms. Additionally, donor sequencescan comprise a vector molecule containing sequences that are nothomologous to the region of interest in cellular chromatin. A donormolecule can contain several, discontinuous regions of homology tocellular chromatin, and, for example, lead to a deletion of a Bcl11aenhancer region (or a fragment thereof) when used as a substrate forrepair of a DBS induced by one of the nucleases described here. Further,for targeted insertion of sequences not normally present in a region ofinterest, said sequences can be present in a donor nucleic acid moleculeand flanked by regions of homology to sequence in the region ofinterest.

Polynucleotides for insertion can also be referred to as “exogenous”polynucleotides, “donor” polynucleotides or molecules or “transgenes.”The donor polynucleotide can be DNA or RNA, single-stranded and/ordouble-stranded and can be introduced into a cell in linear or circularform. See, e.g., U.S. Patent Publication Nos. 2010/0047805 and2011/0207221. The donor sequence(s) are preferably contained within aDNA MC, which may be introduced into the cell in circular or linearform. If introduced in linear form, the ends of the donor sequence canbe protected (e.g., from exonucleolytic degradation) by methods known tothose of skill in the art. For example, one or more dideoxynucleotideresidues are added to the 3′ terminus of a linear molecule and/orself-complementary oligonucleotides are ligated to one or both ends.See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additionalmethods for protecting exogenous polynucleotides from degradationinclude, but are not limited to, addition of terminal amino group(s) andthe use of modified internucleotide linkages such as, for example,phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyriboseresidues. If introduced in double-stranded form, the donor may includeone or more nuclease target sites, for example, nuclease target sitesflanking the transgene to be integrated into the cell's genome. See,e.g., U.S. Patent Publication No. 2013/0326645.

A polynucleotide can be introduced into a cell as part of a vectormolecule having additional sequences such as, for example, replicationorigins, promoters and genes encoding antibiotic resistance. Moreover,donor polynucleotides can be introduced as naked nucleic acid, asnucleic acid complexed with an agent such as a liposome or poloxamer, orcan be delivered by viruses (e.g., adenovirus, AAV, herpesvirus,retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

In certain embodiments, the double-stranded donor includes sequences(e.g., coding sequences, also referred to as transgenes) greater than 1kb in length, for example between 2 and 200 kb, between 2 and 10 kb (orany value therebetween). The double-stranded donor also includes atleast one nuclease target site, for example. In certain embodiments, thedonor includes at least 2 target sites, for example for a pair of ZFNsor TALENs. Typically, the nuclease target sites are outside thetransgene sequences, for example, 5′ and/or 3′ to the transgenesequences, for cleavage of the transgene. The nuclease cleavage site(s)may be for any nuclease(s). In certain embodiments, the nuclease targetsite(s) contained in the double-stranded donor are for the samenuclease(s) used to cleave the endogenous target into which the cleaveddonor is integrated via homology-independent methods.

The donor is generally inserted so that its expression is driven by theendogenous promoter at the integration site, namely the promoter thatdrives expression of the endogenous gene into which the donor isinserted (e.g., globin, AAVS1, etc.). However, it will be apparent thatthe donor may comprise a promoter and/or enhancer, for example aconstitutive promoter or an inducible or tissue specific promoter.

The donor molecule may be inserted into an endogenous gene such thatall, some or none of the endogenous gene is expressed. In otherembodiments, the transgene (e.g., with or without globin encodingsequences) is integrated into any endogenous locus, for example asafe-harbor locus. See, e.g., U.S. Patent Publication Nos. 2008/0299580;2008/0159996 and 2010/00218264.

Furthermore, although not required for expression, exogenous sequencesmay also include transcriptional or translational regulatory sequences,for example, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.

The transgenes carried on the donor sequences described herein may beisolated from plasmids, cells or other sources using standard techniquesknown in the art such as PCR. Donors for use can include varying typesof topology, including circular supercoiled, circular relaxed, linearand the like. Alternatively, they may be chemically synthesized usingstandard oligonucleotide synthesis techniques. In addition, donors maybe methylated or lack methylation. Donors may be in the form ofbacterial or yeast artificial chromosomes (BACs or YACs).

The double-stranded donor polynucleotides described herein may includeone or more non-natural bases and/or backbones. In particular, insertionof a donor molecule with methylated cytosines may be carried out usingthe methods described herein to achieve a state of transcriptionalquiescence in a region of interest.

The exogenous (donor) polynucleotide may comprise any sequence ofinterest (exogenous sequence). Exemplary exogenous sequences include,but are not limited to any polypeptide coding sequence (e.g., cDNAs),promoter sequences, enhancer sequences, epitope tags, marker genes,cleavage enzyme recognition sites and various types of expressionconstructs. Marker genes include, but are not limited to, sequencesencoding proteins that mediate antibiotic resistance (e.g., ampicillinresistance, neomycin resistance, G418 resistance, puromycin resistance),sequences encoding colored or fluorescent or luminescent proteins (e.g.,green fluorescent protein, enhanced green fluorescent protein, redfluorescent protein, luciferase), and proteins which mediate enhancedcell growth and/or gene amplification (e.g., dihydrofolate reductase).Epitope tags include, for example, one or more copies of FLAG, His, myc,Tap, HA or any detectable amino acid sequence.

In a preferred embodiment, the exogenous sequence (transgene) comprisesa polynucleotide encoding any polypeptide of which expression in thecell is desired, including, but not limited to antibodies, antigens,enzymes, receptors (cell surface or nuclear), hormones, lymphokines,cytokines, reporter polypeptides, growth factors, and functionalfragments of any of the above. The coding sequences may be, for example,cDNAs.

For example, the exogenous sequence may comprise a sequence encoding apolypeptide that is lacking or non-functional in the subject having agenetic disease, including but not limited to any of the followinggenetic diseases: achondroplasia, achromatopsia, acid maltasedeficiency, adenosine deaminase deficiency (OMIM No. 102700),adrenoleukodystrophy, aicardi syndrome, alpha-1 antitrypsin deficiency,alpha-thalassemia, androgen insensitivity syndrome, apert syndrome,arrhythmogenic right ventricular, dysplasia, ataxia telangictasia, barthsyndrome, beta-thalassemia, blue rubber bleb nevus syndrome, canavandisease, chronic granulomatous diseases (CGD), cri du chat syndrome,cystic fibrosis, dercum's disease, ectodermal dysplasia, fanconi anemia,fibrodysplasiaossificans progressive, fragile X syndrome, galactosemis,Gaucher's disease, generalized gangliosidoses (e.g., GMI),hemochromatosis, the hemoglobin C mutation in the 6^(th) codon ofbeta-globin (HbC), hemophilia, Huntington's disease, Hurler Syndrome,hypophosphatasia, Klinefleter syndrome, Krabbes Disease, Langer-GiedionSyndrome, leukocyte adhesion deficiency (LAD, OMIM No. 116920),leukodystrophy, long QT syndrome, Marfan syndrome, Moebius syndrome,mucopolysaccharidosis (MPS), nail patella syndrome, nephrogenic diabetesinsipdius, neurofibromatosis, Neimann-Pick disease, osteogenesisimperfecta, porphyria, Prader-Willi syndrome, progeria, Proteussyndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome,Sanfilippo syndrome, severe combined immunodeficiency (SCID), Shwachmansyndrome, sickle cell disease (sickle cell anemia), Smith-Magenissyndrome, Stickler syndrome, Tay-Sachs disease, Thrombocytopenia AbsentRadius (TAR) syndrome, Treacher Collins syndrome, trisomy, tuberoussclerosis, Turner's syndrome, urea cycle disorder, von Hippel-Landaudisease, Waardenburg syndrome, Williams syndrome, Wilson's disease,Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome (XLP,OMIM No. 308240).

Additional exemplary diseases that can be treated by targetedintegration include acquired immunodeficiencies, lysosomal storagediseases (e.g., Gaucher's disease, GM1, Fabry disease and Tay-Sachsdisease), mucopolysaccahidosis (e.g. Hunter's disease, Hurler'sdisease), hemoglobinopathies (e.g., sickle cell diseases, HbC,α-thalassemia, β-thalassemia) and hemophilias.

In certain embodiments, the exogenous sequences can comprise a markergene (described above), allowing selection of cells that have undergonetargeted integration, and a linked sequence encoding an additionalfunctionality. Non-limiting examples of marker genes include GFP, drugselection marker(s) and the like.

Additional gene sequences that can be inserted may include, for example,wild-type genes to replace mutated sequences. For example, a wild-typeFactor IX gene sequence may be inserted into the genome of a stem cellin which the endogenous copy of the gene is mutated. The wild-type copymay be inserted at the endogenous locus, or may alternatively betargeted to a safe harbor locus.

Construction of such expression cassettes, following the teachings ofthe present specification, utilizes methodologies well known in the artof molecular biology (see, for example, Ausubel or Maniatis). Before useof the expression cassette to generate a transgenic animal, theresponsiveness of the expression cassette to the stress-inducerassociated with selected control elements can be tested by introducingthe expression cassette into a suitable cell line (e.g., primary cells,transformed cells, or immortalized cell lines).

Furthermore, although not required for expression, exogenous sequencesmay also transcriptional or translational regulatory sequences, forexample, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.Further, the control elements of the genes of interest can be operablylinked to reporter genes to create chimeric genes (e.g., reporterexpression cassettes).

Targeted insertion of non-coding nucleic acid sequence may also beachieved. Sequences encoding antisense RNAs, RNAi, shRNAs and micro RNAs(miRNAs) may also be used for targeted insertions.

In additional embodiments, the donor nucleic acid may comprisenon-coding sequences that are specific target sites for additionalnuclease designs. Subsequently, additional nucleases may be expressed incells such that the original donor molecule is cleaved and modified byinsertion of another donor molecule of interest. In this way,reiterative integrations of donor molecules may be generated allowingfor trait stacking at a particular locus of interest or at a safe harborlocus.

Delivery

The nucleases as described herein (Table 1), polynucleotides encodingthese nucleases, donor polynucleotides and compositions comprising theproteins and/or polynucleotides described herein may be delivered invivo or ex vivo by any suitable means into any cell type.

Suitable cells include eukaryotic (e.g., animal) and prokaryotic cellsand/or cell lines. Non-limiting examples of such cells or cell linesgenerated from such cells include COS, CHO (e.g., CHO—S, CHO-K1,CHO-DG44, CHO-DUXBI1, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79,B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F,HEK293-H, HEK293-T), and perC6 cells as well as insect cells such asSpodopterafugiperda (Sf), or fungal cells such as Saccharomyces, Pichiaand Schizosaccharomyces. In certain embodiments, the cell line is a CHO,MDCK or HEK293 cell line. Suitable cells also include stem cells suchas, by way of example, embryonic stem cells, induced pluripotent stemcells, hematopoietic stem cells, neuronal stem cells and mesenchymalstem cells.

Methods of delivering nucleases as described herein are described, forexample, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692;6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219 and7,163,824, the disclosures of all of which are incorporated by referenceherein in their entireties.

Nucleases and/or donor constructs as described herein may also bedelivered using vectors containing sequences encoding one or more of theZFN(s), described herein. Any vector systems may be used including, butnot limited to, plasmid vectors, retroviral vectors, lentiviral vectors,adenovirus vectors, poxvirus vectors; herpesvirus vectors andadeno-associated virus vectors, etc. See, also, U.S. Pat. Nos.6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219 and7,163,824, incorporated by reference herein in their entireties.Furthermore, it will be apparent that any of these vectors may compriseone or more of the sequences needed for treatment. Thus, when one ormore nucleases and a donor construct are introduced into the cell, thenucleases and/or donor polynucleotide may be carried on the same vectoror on different vectors (DNA MC(s)). When multiple vectors are used,each vector may comprise a sequence encoding one or multiple nucleasesand/or donor constructs. Conventional viral and non-viral based genetransfer methods can be used to introduce nucleic acids encodingnucleases and/or donor constructs in cells (e.g., mammalian cells) andtarget tissues. Non-viral vector delivery systems include DNA or RNAplasmids, DNA MCs, naked nucleic acid, and nucleic acid complexed with adelivery vehicle such as a liposome or poloxamer. Suitable non-viralvectors include nanotaxis vectors, including vectors commerciallyavailable from InCellArt (France). Viral vector delivery systems includeDNA and RNA viruses, which have either episomal or integrated genomesafter delivery to the cell. For a review of in vivo delivery ofengineered DNA-binding proteins and fusion proteins comprising thesebinding proteins, see, e.g., Rebar (2004) Expert Opinion Invest. Drugs13(7):829-839; Rossi et al. (2007) Nature Biotech. 25(12):1444-1454 aswell as general gene delivery references such as Anderson (1992) Science256:808-813; Nabel & Felgner (1993) TIBTECH 11:211-217; Mitani & Caskey(1993) TIBTECH 11:162-166; Dillon (1993) TIBTECH 11:167-175; Miller(1992) Nature 357:455-460; Van Brunt (1988) Biotechnology6(10):1149-1154; Vigne (1995) Restorative Neurology and Neuroscience8:35-36; Kremer & Perricaudet (1995) British Medical Bulletin51(1):31-44; Haddada et al., in Current Topics in Microbiology andImmunology Doerfler and Böhm (eds.) (1995); and Yu et al. (1994) GeneTherapy 1:13-26.

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Sonoporationusing, e.g., the Sonitron 2000 system (Rich-Mar) can also be used fordelivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc., (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, International PatentPublication Nos. WO 91/17424 and WO 91/16024.

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal (1995) Science 270:404-410; Blaese et al.(1995) Cancer Gene Ther. 2:291-297; Behr et al. (1994) BioconjugateChem. 5:382-389; Remy et al. (1994) Bioconjugate Chem. 5:647-654; Gao etal. (1995) Gene Therapy 2:710-722; Ahmad et al. (1992) Cancer Res.52:4817-4820; U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975;4,485,054; 4,501,728; 4,774,085; 4,837,028 and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet al. (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs, TALEs and/or CRISPR/Cas systems takeadvantage of highly evolved processes for targeting a virus to specificcells in the body and trafficking the viral payload to the nucleus.Viral vectors can be administered directly to patients (in vivo) or theycan be used to treat cells in vitro and the modified cells areadministered to patients (ex vivo). Conventional viral based systems forthe delivery of ZFPs include, but are not limited to, retroviral,lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplexvirus vectors for gene transfer. Integration in the host genome ispossible with the retrovirus, lentivirus, and adeno-associated virusgene transfer methods, often resulting in long term expression of theinserted transgene. Additionally, high transduction efficiencies havebeen observed in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al. (1992) J. Virol.66:2731-2739; Johann et al. (1992) J. Virol. 66:1635-1640; Sommerfelt etal. (1990) Virol. 176:58-59; Wilson et al. (1989) J. Virol.63:2374-2378; Miller et al. (1991) J. Virol. 65:2220-2224; InternationalPatent Publication No. WO 94/26877).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al. (1987) Virology160:38-47; U.S. Pat. No. 4,797,368; International Patent Publication No.WO 93/24641; Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994)J. Clin. Invest. 94:1351. Construction of recombinant AAV vectors aredescribed in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260;Tratschin, et al. (1984) Mol. Cell. Biol. 4:2072-2081; Hermonat &Muzyczka (1984) PNAS 81:6466-6470; and Samulski et al. (1989) J. Virol.63:03822-3828.

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al. (1995) Blood 85:3048-305; Kohn et al.(1995) Nat. Med. 1:1017-102; Malech et al. (1997) PNAS94(22):12133-12138). PA317/pLASN was the first therapeutic vector usedin a gene therapy trial. (Blaese et al. (1995) Science 270:475-480).Transduction efficiencies of 50% or greater have been observed for MFG-Spackaged vectors. (Ellem et al. (1997) Immunol Immunother. 44(1):10-20;Dranoff et al. (1997) Hum. Gene Ther. 1:111-2.

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al. (1998) Lancet 351(9117):1702-3, Kearns et al. (1996) GeneTher. 9:748-55). Other AAV serotypes, including AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9 and AAVrh.10 and any novel AAV serotype canalso be used in accordance with the present invention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al. (1998) Hum.Gene Ther. 7:1083-9). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.(1996) Infection 24(1):5-10; Sterman et al. (1998) Hum. Gene Ther.9(7):1083-1089; Welsh et al. (1995) Hum. Gene Ther. 2:205-18; Alvarez etal. (1997) Hum. Gene Ther. 5:597-613; Topf et al. (1998) Gene Ther.5:507-513; Sterman et al. (1998) Hum. Gene Ther. 7:1083-1089.

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al. (1995) Proc. Natl.Acad. Sci. USA 92:9747-9751, reported that Moloney murine leukemia viruscan be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingnucleases and/or donor constructs can also be administered directly toan organism for transduction of cells in vivo. Alternatively, naked DNAcan be administered. Administration is by any of the routes normallyused for introducing a molecule into ultimate contact with blood ortissue cells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Vectors suitable for introduction of polynucleotides (e.g.nuclease-encoding and/or double-stranded donors) described hereininclude non-integrating lentivirus vectors (IDLV). See, for example, Oryet al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222; U.S.Patent Publication No 2009/0117617.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

It will be apparent that the nuclease-encoding sequences and donorconstructs can be delivered using the same or different systems. Forexample, the nucleases and donors can be carried by the same DNA MC.Alternatively, a donor polynucleotide can be carried by a MC, while theone or more nucleases can be carried by a standard plasmid or AAVvector. Furthermore, the different vectors can be administered by thesame or different routes (intramuscular injection, tail vein injection,other intravenous injection, intraperitoneal administration and/orintramuscular injection. The vectors can be delivered simultaneously orin any sequential order.

Thus, the instant disclosure includes in vivo or ex vivo treatment ofdiseases and conditions that are amenable to insertion of a transgenesencoding a therapeutic protein. The compositions are administered to ahuman patient in an amount effective to obtain the desired concentrationof the therapeutic polypeptide in the serum or the target organ orcells. Administration can be by any means in which the polynucleotidesare delivered to the desired target cells. For example, both in vivo andex vivo methods are contemplated. Intravenous injection to the portalvein is a preferred method of administration. Other in vivoadministration modes include, for example, direct injection into thelobes of the liver or the biliary duct and intravenous injection distalto the liver, including through the bepatic artery, direct injection into the liver parenchyma, injection via the hepatic artery, and/orretrograde injection through the biliary tree. Ex vivo modes ofadministration include transduction in vitro of resected hepatocytes orother cells of the liver, followed by infusion of the transduced,resected hepatocytes back into the portal vasculature, liver parenchymaor biliary tree of the human patient, see e.g., Grossman et al. (1994)Nature Genetics 6:335-341.

The effective amount of nuclease(s) and donor to be administered willvary from patient to patient and according to the therapeuticpolypeptide of interest. Accordingly, effective amounts are bestdetermined by the physician administering the compositions andappropriate dosages can be determined readily by one of ordinary skillin the art. After allowing sufficient time for integration andexpression (typically 4-15 days, for example), analysis of the serum orother tissue levels of the therapeutic polypeptide and comparison to theinitial level prior to administration will determine whether the amountbeing administered is too low, within the right range or too high.Suitable regimes for initial and subsequent administrations are alsovariable, but are typified by an initial administration followed bysubsequent administrations if necessary. Subsequent administrations maybe administered at variable intervals, ranging from daily to annually toevery several years. One of skill in the art will appreciate thatappropriate immunosuppressive techniques may be recommended to avoidinhibition or blockage of transduction by immunosuppression of thedelivery vectors, see e.g., Vilquin et al. (1995) Human Gene Ther.6:1391-1401.

Formulations for both ex vivo and in vivo administrations includesuspensions in liquid or emulsified liquids. The active ingredientsoften are mixed with excipients which are pharmaceutically acceptableand compatible with the active ingredient. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like, andcombinations thereof. In addition, the composition may contain minoramounts of auxiliary substances, such as, wetting or emulsifying agents,pH buffering agents, stabilizing agents or other reagents that enhancethe effectiveness of the pharmaceutical composition.

Cells

Also described herein are cells and/or cell lines in which an endogenousBCL11A enhancer sequence is modified by the nucleases described herein(Table 1). The modification may be, for example, as compared to thewild-type sequence of the cell. The cell or cell lines may beheterozygous or homozygous for the modification. The modifications tothe BCL11A sequence may comprise insertions, deletions and/orcombinations thereof.

The modification is preferably at or near the nuclease(s) binding and/orcleavage site(s), for example, within 1-300 (or any value therebetween)base pairs upstream or downstream of the site(s) of cleavage, morepreferably within 1-100 base pairs (or any value therebetween) of eitherside of the binding and/or cleavage site(s), even more preferably within1 to 50 base pairs (or any value therebetween) on either side of thebinding and/or cleavage site(s). In certain embodiments, themodification is at or near the “+58” region of the BCL11A enhancer, forexample, at or near a nuclease binding site shown in any of the firstcolumn of Table 1.

Any cell or cell line may be modified, for example a stem cell, forexample an embryonic stem cell, an induced pluripotent stem cell, ahematopoietic stem cell, a neuronal stem cell and a mesenchymal stemcell. Other non-limiting examples of cells as described herein includeT-cells (e.g., CD4+, CD3+, CD8+, etc.); dendritic cells; B-cells. Adescendent of a stem cell, including a partially or fully differentiatedcell, is also provided (e.g., a RBC or RBC precursor cell). Non-limitingexamples other cell lines including a modified BCL11A sequence includeCOS, CHO (e.g., CHO—S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV),VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa,HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells as well asinsect cells such as Spodopterafugiperda (Sf), or fungal cells such asSaccharomyces, Pichia and Schizosaccharomyces.

The cells as described herein are useful in treating and/or preventing adisorder, for example, by ex vivo therapies. The nuclease-modified cellscan be expanded and then reintroduced into the patient using standardtechniques. See, e.g., Tebas et al (2014) New Eng J Med 370(10):901. Inthe case of stem cells, after infusion into the subject, in vivodifferentiation of these precursors into cells expressing the functionaltransgene also occurs. Pharmaceutical compositions comprising the cellsas described herein are also provided. In addition, the cells may becryopreserved prior to administration to a patient.

Any of the modified cells or cell lines disclosed herein may showincreased expression of gamma globin. Compositions such aspharmaceutical compositions comprising the genetically modified cells asdescribed herein are also provided

Applications

The methods and compositions disclosed herein are for modifyingexpression of protein, or correcting an aberrant gene sequence thatencodes a protein expressed in a genetic disease, such as a sickle celldisease or a thalassemia. Thus, the methods and compositions provide forthe treatment and/or prevention of such genetic diseases. Genomeediting, for example of stem cells, can be used to correct an aberrantgene, insert a wild type gene, or change the expression of an endogenousgene. By way of non-limiting example, a wild type gene, e.g. encoding atleast one globin (e.g., α and/or β globin), may be inserted into a cell(e.g., into an endogenous BCL11a enhancer sequence using one or morenucleases as described herein) to provide the globin proteins deficientand/or lacking in the cell and thereby treat a genetic disease, e.g., ahemoglobinopathy, caused by faulty globin expression. Alternatively orin addition, genomic editing with or without administration of theappropriate donor, can correct the faulty endogenous gene, e.g.,correcting the point mutation in α- or β-hemoglobin, to restoreexpression of the gene and/or treat a genetic disease, e.g. sickle celldisease and/or knock out or alteration (overexpression or repression) ofany direct or indirect globin regulatory gene (e.g. inactivation of theγ globin-regulating gene BCL11A or the BCL11A-regulator KLF1).Specifically, the methods and compositions of the invention have use inthe treatment or prevention of hemoglobinopathies.

The nucleases of the invention are targeted to the BCL11A enhancerregion, known to be required for the expression of BCL11A, and hence thedown regulation of gamma globin expression. Modification of thisenhancer region may result in erythrocytes with increased gamma globinexpression, and thus may be helpful for the treatment or prevention ofsickle cell disease or beta thalassemia.

The following Examples relate to exemplary embodiments of the presentdisclosure in which the nuclease comprises a zinc finger nuclease (ZFN).It will be appreciated that this is for purposes of exemplification onlyand that other nucleases can be used, for example TtAgo and CRISPR/Cassystems, homing endonucleases (meganucleases) with engineeredDNA-binding domains and/or fusions of naturally occurring of engineeredhoming endonucleases (meganucleases) DNA-binding domains andheterologous cleavage domains and/or fusions of meganucleases and TALEproteins.

EXAMPLES Example 1: Assembly of Zinc Finger Nucleases

ZFNs were assembled against the human BCL11A gene and were tested byCEL1 assays as described in Miller et al. (2007) Nat. Biotechnol.25:778-785. ZFNs specific for the +58 region of the enhancer region weremade as described. The nucleases are shown below in Table 1:

TABLE 1 ZFN pairs specific for +58 BCL11A enhancer region SBS # (targetsite, Design 5′-3′) F1 F2 F3 F4 F5 F6 Left partner 46801 DQSNLRA RPYTLRLSGYNLEN TSGSLTR DQSNLRA AQCCLFH aaAGCAACtG (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID TTAGCTTGCA NO: 2) NO: 3) NO: 4) NO: 5) NO: 2)NO: 6) Ctagacta (SEQ ID NO: 1) 51446 STGNLTN TSGSLTR DQSNLRA AQCCLFH N/AN/A aaAGCAACtG (SEQ ID (SEQ ID (SEQ ID (SEQ ID TTAGCttgca NO: 7) NO: 5)NO: 2) NO: 6) ctagacta (SEQ ID NO: 1) 51463 STGNLTN TSGSLTR DQSNLRAAQCCLFH N/A N/A aaAGCAACtG (SEQ ID (SEQ ID (SEQ ID (SEQ ID TTAGCttgcaNO: 7) NO: 5) NO: 2) NO: 6) ctagacta (SEQ ID NO: 1) 51484 DQSNLRARPYTLRL SRGALKT TSGSLTR DQSNLRA AQCCLFH aaAGCAACtG (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID TTAGCTTGCA NO: 2) NO: 3) NO: 8) NO: 5)NO: 2) NO: 6) Ctagacta (SEQ ID NO: 1) 51856 DQSNLRA RNFSLTM SNGNLRNTSGSLTR DQSNLRA AQCCLFH aaAGCAACtG (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID TTAGCTTGCA NO: 2) NO: 9) NO: 10) NO: 5) NO: 2) NO: 6)Ctagacta (SEQ ID NO: 1) 51857 DQSNLRA RNFSLTM STGNLTN TSGSLTR DQSNLRAAQCCLFH aaAGCAACtG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDTTAGCTTGCA NO: 2) NO: 9) NO: 7) NO: 5) NO: 2) NO: 6) Ctagacta (SEQ IDNO: 1) 51862 DQSNLRA RNFSLTM SSYNLAN TSGSLTR DQSNLRA AQCCLFH aaAGCAACtG(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID TTAGCTTGCA NO: 2) NO: 9)NO: 11) NO: 5) NO: 2) NO: 6) Ctagacta (SEQ ID NO: 1) 51477 DQSNLRARPYTLRL SSSNLTN TSGSLTR DQSNLRA AQCCLFH aaAGCAACtG (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID TTAGCTTGCA NO: 2) NO: 3) NO: 26) NO: 5)NO: 2) NO: 6) Ctagacta (SEQ ID NO: 1) 51478 DQSNLRA RPYTLRL SSSNLGNTSGSLTR DQSNLRA AQCCLFH aaAGCAACtG (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID TTAGCTTGCA NO: 2) NO: 3) NO: 27) NO: 5) NO: 2) NO: 6)Ctagacta (SEQ ID NO: 1) 51487 DQSNLRA RPYTLRL SRSALRV TSGSLTR DQSNLRAAQCCLFH aaAGCAACtG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDTTAGCTTGCA NO: 2) NO: 3) NO: 28) NO: 5) NO: 2) NO: 6) Ctagacta (SEQ IDNO: 1) Right Partner 47923 RSDHLTQ QSGHLAR QKGTLGE QSSDLSR RRDNLHS N/AcaCAGGCTCC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGGAAGGgtt NO: 13)NO: 14) NO: 15) NO: 16) NO: 17) tggcctct (SEQ ID NO: 12) 51536 RSDHLTQQSGHLAR QKGTLGE RHRDLSR RRDNLHS N/A caCAGGCTCC (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID AGGAAGGgtt NO: 13) NO: 14) NO: 15) NO: 18) NO: 17)tggcctct (SEQ ID NO: 12) 51949 RNDHRTT QKAHLIR QKGTLGE RGRDLSR RRDNLHSN/A caCAGGCTCC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGGAAGGgttNO: 19) NO: 20) NO: 15) NO: 21) NO: 17) tggcctct (SEQ ID NO: 12) 51990RSDHLTQ QRAHLTR QKGTLGE HRNTLVR RRDNLHS N/A caCAGGCTCC (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID AGGAAGGgtt NO: 13) NO: 22) NO: 15) NO: 23)NO: 17) tggcctct (SEQ ID NO: 12) 51993 RSDHLTQ QRAHLTR QSGTRNH HRNTLVRRRDNLHS N/A caCAGGCTCC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDAGGAAGGgtt NO: 13) NO: 22) NO: 24) NO: 23) NO: 17) tggcctct (SEQ IDNO: 12) 51979 RSDHLTQ QKAHLIR QKGTLGE RGRDLSR RRDNLHS N/A caCAGGCTCC(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGGAAGGgtt NO: 13) NO: 20)NO: 15) NO: 21) NO: 17) tggcctct (SEQ ID NO: 12) 51982 RSDHLTQ QKAHLIRQSGTRNH RGRDLSR RRDNLHS N/A caCAGGCTCC (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID AGGAAGGgtt NO: 13) NO: 20) NO: 24) NO: 21) NO: 17) tggcctct(SEQ ID NO: 12) 52015 RNDHRTT QKAHLIR QKGTLGE LKRTLKR RRDNLHS N/AcaCAGGCTCC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGGAAGGgtt NO: 19)NO: 20) NO: 15) NO: 25) NO: 17) tggcctct (SEQ ID NO: 12) 52032 RSDHLTQQSGHLAR QSGTRNH QSSDLSR RRDNLHS N/A caCAGGCTCC (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID AGGAAGGgtt NO: 13) NO: 14) NO: 24) NO: 16) NO: 17)tggcctct (SEQ ID NO: 12) 51541 RSDHLTQ QSGHLAR QKGTLGE RHRDLSR RRDNLHSN/A caCAGGCTCC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGGAAGGgttNO: 13) NO: 14) NO: 15) NO: 18) NO: 17) tggcctct (SEQ ID NO: 12) 51519RSDHLTQ QSGHLAR QSGTRNH QSSDLSR RRDNLHS N/A caCAGGCTCC (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID AGGAAGGgtt NO: 13) NO: 14) NO: 24) NO: 16)NO: 17) tggcctct (SEQ ID NO: 12) 51534 RSDHLTQ QSGHLAR QKGTLGE RGRDLSRRRDNLHS N/A caCAGGCTCC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDAGGAAGGgtt NO: 13) NO: 14) NO: 15) NO: 21) NO: 17) tggcctct (SEQ IDNO: 12) 51535 RSDHLTQ QSGHLAR QKGTLGE RSRDLTR RRDNLHS N/A caCAGGCTCC(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGGAAGGgtt NO: 13) NO: 14)NO: 15) NO: 29) NO: 17) tggcctct (SEQ ID NO: 12) 51556 RSDHLTQ QSGHLARQKGTLGE FRQTRAR RRDNLHS N/A caCAGGCTCC (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID AGGAAGGgtt NO: 13) NO: 14) NO: 15) NO: 30) NO: 17) tggcctct(SEQ ID NO: 12) *51446 and 51463 differ in linker sequences

All ZFNs were tested for functionality (cleavage activity) and found tobe active.

Example 2: Activity of ZFN in Human K562 Cells

Briefly, human K562 cells were cultured in RPMI supplemented with 10%FBS and 200,000 cells were transfected with a suboptimal concentrationof 25 ng of each of the plasmid DNA encoding the left and right ZFNpartners by Amaxa Nucleofector® following the manufacturer'sinstructions (Table 2a). In addition, the experiments were performedwith 25 ng of the left ZFN and 5 ng of the right ZFN (Table 2b). TheCel-I assay (Surveyor™, Transgenomics) as described in Perez et al.(2008) Nat. Biotechnol. 26:808-816 and Guschin et al. (2010) Methods MolBiol. 649:247-56), was used to detect ZFN-induced modifications of thetarget gene two or three days after transfection. In this assay,PCR-amplification of the target site was followed by quantification ofinsertions and/or deletions (“indels”) by sequencing. Deep sequencing onthe Illumina platform (“miSEQ”) was used according to the manufacturer'sinstructions to measure editing efficiency as well as nature ofediting-generated alleles. The results are shown below in Table 2, wherethe numbers indicate the percent NHEJ activity observed:

TABLE 2a Matrix screen in K562 cells (25 ng each ZFN) 51949 51977 5197951982 51990 51993 52015 52032 ave. 51856 22.2 21.0 27.7 23.2 21.0 23.917.0 26.7 22.8 51857 28.5 24.4 29.4 28.1 26.7 23.9 19.2 32.8 26.6 5186215.9 16.3 15.5 15.3 11.7 20.8 13.9 22.4 16.5 51877 12.0 13.2 12.4 13.910.7 11.3 9.1 13.8 12.1 51879 14.2 15.1 14.9 13.8 12.0 14.4 10.8 16.814.0 average 18.6 18.0 20.0 18.8 16.4 18.9 14.0 22.5 46801: 47923 9.3

TABLE 2b Matrix screen in K562 cells (25 ng left ZFN, 5 ng right ZFN)51949 51977 51979 51982 51990 51993 52015 52032 ave 51856 10.9 12.6 16.913.4 11.0 12.4 8.5 15.5 12.7 51857 15.7 12.8 15.2 14.1 12.5 14.4 11.814.5 13.9 51862 13.0 11.4 13.9 10.4 14.3 12.2 12.2 14.3 12.7 51877 8.27.2 8.8 6.7 8.2 7.1 7.0 6.9 7.5 51879 8.5 7.9 11.5 8.3 8.4 7.1 4.9 11.28.5 average 11.3 10.4 13.3 10.6 10.9 10.6 8.9 12.5 46801: 47923 7.3

The ZFNs were also constructed using four different linkers between theDNA binding domain and the nuclease domain (see U.S. Patent PublicationNo. 2015/0064789). The linker sequences tested are shown below, wherethe ‘HTKIH’ portion of the sequence is the carboxy terminus of the DNAbinding domain and the ‘ELEEK’ portion is the amino terminus of thenuclease domain. The underlined portion is the linker sequence betweenthe two domains:

Linker Sequences

L7a: (SEQ ID NO: 31) HTKIH LRGSQLVKSKSEAAAR ELEEK L7c5: (SEQ ID NO: 32)HTKIH LRGSISRARPLNPHP ELEEK L0: (SEQ ID NO: 33)HTKIH LRGSISRARPLNPHP ELEEK L8c4: (SEQ ID NO: 34)HTKIH LRGSYAPMPPLALASP ELEEK

In these experiments, the L0 or L8c4 linkers were tested on the rightside partner in combination with the L7c5 or L7a linkers on the leftside partner. The combinations of the ZFNs were tested for cleavageactivity in K562 cells, and the results (percent NHEJ activity) areshown below in Table 3.

TABLE 3 ZEN activity varying domain linkers L7c5 L7c5 L7c5 L7a L7a SBS51856 51857 51862 46801 51446 ave L0 51949 6.4 9.2 7.3 4.8 10.6 7.66 L051977 8.8 8.9 5.6 5.0 8.7 7.41 L0 51979 11.4 10.5 10.8 4.5 11.3 9.70 L051982 8.3 9.7 9.3 4.9 10.5 8.56 L0 51990 10.3 11.0 7.5 5.3 10.2 8.86 L051993 10.7 13.5 7.3 5.1 10.9 9.52 L0 52015 8.5 8.6 7.2 5.7 6.7 7.34 L052032 11.5 11.3 7.0 5.0 14.8 9.91 L0 47923 7.6 7.6 5.0 4.0 11.5 7.14L8c4 52075 10.9 10.9 7.2 5.6 11.7 9.27 L8c4 52103 9.9 8.9 8.5 3.8 10.98.37 L8c4 52105 16.7 13.0 16.3 6.3 15.2 13.49 L8c4 52108 9.7 7.5 6.5 4.212.0 7.99 L8c4 52116 12.0 15.7 9.8 4.0 13.4 10.99 L8c4 52119 13.1 9.88.3 4.5 10.3 9.19 L8c4 52141 15.2 7.2 6.9 3.4 13.2 9.17 L8c4 52158 9.310.3 7.4 3.6 12.5 8.64 L8c4 51536 13.3 12.1 11.3 8.0 15.4 12.04 ave−>10.76 10.32 8.28 4.88 11.65

Example 3: Activity of the ZFNs in CD34+ Cells

ZFNs as described herein were also tested in human CD34+ cells. For theCD34+ transduction, a BTX ECM830 device with a 2 mm gap cuvette wasused. Human CD34+ cells were grown in x-vivo10 media (Lonza) with1×CC110 (Stem cell Technology) in non-tissue culture treated plates. Thecells were counted and collected by centrifugation at 1200 rpm for 10minutes at room temperature. The cells were washed 1-2× with roomtemperature PBS. 200,000 cells were used for each transfection, and theywere resuspended in 100 μL BTexpress solution. For the CD34+experiments, RNAs encoding the ZFNs was used rather than DNA. RNA wasgenerated using a mMessageMachine T7 Ultra Kit (Ambion). 500 ng of RNAencoding each ZFN was added per transfection and the mixture wastransferred to the cuvette. Immediately following transfer, the mixturewas electroporated at 250V for 5 msec. Pre-warmed media was added to thecuvette and the media plus cells were transferred to a 48 wellnon-tissue culture treated plates and then incubated at 37° C.

After two or three days, the cells were then were subject to genomeanalysis using an Ilumina MiSeq. To quantitate the percent of editedalleles, the genomic region of interest was PCR amplified using primerswhich add the standard Illumina sequencing adapter sequences. A secondgroup of 13 rounds of PCR was performed to add barcode and bridgeadapter sequences to both ends. Sequencing was performed on an IlluminaMiSeq according to manufacturer's protocols for amplicon sequencing. TheMiSeq generates paired-end reads, which are merged and adapter-trimmedusing a standard alignment software. Reads were then demultiplexed bysample via barcode sequence pairs using custom scripts. Ampliconsequences were then globally aligned to a reference sequence via animplementation of the Needleman-Wunsch algorithm (Needlemanand Wunsch(1970) Jour Mol Bio 48(3):443-53). Gaps or insertions in the alignmentwere counted as % NHEJ events, and compared to an untreated controlsample sequence to determine sequence-specific background rates. Theresults are shown below in Table 4.

TABLE 4 ZFN activity in CD34+ cells 51949 51977 51979 51982 51990 5199352015 52032 51536 ave 51856 38.4 11.9 26.9 23.9 37.3 37.0 26.4 32.1 nd29.2 51857 52.1 17.2 39.8 30.9 51.0 43.9 35.2 53.9 40.0 40.4 51862 18.45.2 13.0 12.4 26.6 20.7 13.7 16.2 17.7 16.0 51877 19.6 6.0 8.9 9.6 24.018.3 11.6 19.4 13.7 14.6 51879 36.4 10.6 21.8 19.5 39.2 30.7 21.8 34.422.7 26.4 51446 60.1 22.6 41.2 43.6 57.8 50.4 39.7 47.6 nd 45.4 ave 37.512.3 25.3 23.3 39.3 33.5 24.7 33.9 23.5

When an increased amount of input mRNA was used for a selected set ofrepresentative pairs (1 μg each ZFN), additional amounts of cutting wasobserved, as shown in Table 5.

TABLE 5A Table 5A and 5B: Increasing ZFN concentration leads toincreased activity 0.5 ug: 0.5 ug 46801: 47923 1.6 51556: 51484 17.6 1ug: 1 ug 46801: 47923 1.5 51536: 51446 49.0 52032: 51446 65.7 52032:51857 60.8 51979: 51446 60.0 51979: 51857 47.5 51536: 51857 55.7 GFP noseq

TABLE 5B 51536 51541 51556 51519 51534 51535 51446 83.72 87.79 74.1181.52 84.21 82.22 51484 75.21 84.34 64.20 67.88 79.50 74.22 51463 82.7185.24 74.00 78.81 85.53 nd 51477 72.02 85.11 63.74 70.42 80.96 78.3051478 72.33 82.38 58.17 66.22 75.20 70.90 51487 66.27 83.26 64.11 61.9768.99 70.42

The above transfections were mostly performed under conditionsnon-saturating mRNA inputs to allow us to best compare the activity ofvarious ZFN combinations. To test the maximum amount of modificationobtainable at or near saturating inputs of mRNAs we transfectedincreasing amounts of 2a constructs or combining two ZFNs in one mRNAinto CD34+ cells using BTX electroporation, and the results are shownbelow in Table 6.

TABLE 6 Modification of the Bcl11a Enhancer with Increasing mRNA input2A: 2000 ng 4000 ng 8000 ng 51463_2a_51949 59.60 79.83 82.6951463_2a_52990 67.98 80.17 77.30 51463_2a_52032 58.58 80.36 83.2651857_2a_51949 61.22 73.89 78.26 51857_2a_51990 60.63 70.55 74.4051857_2a_52032 55.84 71.17 74.29 euf_51446_2a_51536 56.36 66.69 74.85euf_46801_2a_47923 51.15 72.20 75.97

The data in Table 6 show very high modification of the ZFN target regionwith increasing mRNA input for all combinations tested.

Similar to the experiments done in Experiment 2 analyzing activity withZFNs comprising varied linkers, the effect of linkers on activity wasalso tested in CD34+ cells. As above, varying amounts of mRNA (either500, 1000 or 2000 ng of each) was used to deliver the ZFNs in theseexperiments. Table 7 shows the effect of linker identity on the activityof the ZFN pairs.

TABLE 7 Effect of linker identity on ZFN activity in CD34+ cells 500 ng51463 51857 1000 ng 51463 51857 2000 ng 51463 51857 L0 51949 29.9 31.051949 48.8 46.7 51949 44.5 43.5 51979 26.3 23.8 51979 39.2 40.0 5197936.3 35.1 51990 28.6 28.1 51990 39.8 41.8 51990 36.1 36.3 52032 24.725.5 52032 39.6 39.7 52032 37.2 33.6 L8c4 51536 23.0 19.1 51536 36.031.0 51536 35.7 30.6 52075 22.1 21.2 52075 34.7 32.0 52075 35.2 27.552105 21.9 19.1 52105 37.2 32.8 52105 36.2 31.6 52116 14.7 13.9 5211627.6 28.8 52116 28.4 28.9 52158 22.2 21.1 52158 36.3 34.9 52158 41.738.2 500 ng 51446 1000 ng 51446 2000 ng 51446 51536 19.8 51536 35.551536 28.7

Example 4: Differentiation of Edited CD34+ Cells and HemoglobinExpression

To test the effect on relative gamma globin expression, the mRNAsencoding a representative sample of the ZFN pairs were introduced intoCD34+ cells (obtained from healthy donor volunteers) by BTXnucleofection according to manufacturer's instructions. The cells werethen differentiated into erythrocytes. Briefly, CD34⁺ cells werepurified using Ficoll-Paque (GE Healthcare) and CD34⁺ microbeads(Miltenyi Biotec) according to the manufacturers' instructions. CD34⁺cells were cultured in Iscove's MDM with BIT 95000 (StemCellTechnologies) in the presence of growth factors. Cells weredifferentiated toward the erythroid lineage using a 3 step liquidculture model. During the first 6 days (first phase), CD34⁺ cells wereexpanded with SCF (100 ng/ml), Flt3-L (100 ng/ml), and IL-3 (20 ng/ml).Expanded cells were then committed and differentiated toward theerythroid lineage (second phase) with Epo (2 U/ml) and SCF (50 ng/ml).See, Giarratana et al. (2011) Blood 118(19):5071-9.

To analyze relative gamma globin expression, the ratios of mRNAsencoding gamma globin, alpha globin and beta globin following ZFNtreatment were determined at 14 days after the start of differentiationby RT-PCR analysis. The analysis was done by standard Taqman® analysis,following the protocol and using gene specific assays supplied by themanufacturer (Applied Biosystems). The relative levels of gamma globin(HBG) was normalized by the level of alpha (HBA) or beta globin (HBB)expression where the ratio was compared to the gamma/alpha or gamma/betaratio in control cells.

The data are presented below in Table 8, and demonstrate that incomparison with cells that were treated with the GFP encoding plasmid,there was an increase in gamma globin expression in ZFN-treated cells.

TABLE 8 Change in gamma globin expression relative to alpha or betaglobin in edited CD34+ cells ZFN pair HBG/HBA HBG/HBB 46801/47293 2.74.4 51446/51536 5.0 8.1 51463/51536 8.0 10.5 51484/51536 3.8 6.4 GFP 1.62.8

In vitro erythroid differentiation of the CD34+ cells transfected at ornear saturating mRNA concentrations followed by RT-PCR analysis ofglobin expression as described above shows very efficient gamma-globinactivation by all selected ZFN pairs targeting the Bcl11a enhancer whencompared to the GFP mRNA transfected control sample (FIG. 1). The datais presented below in Table 9.

TABLE 9 Increased ratio of human gamma globin expression HBG/HBB mRNAused 2 μg 4 μg 8 μg 51463-2a-51949 10.33 17.56 21.84 51463-2a-5199012.75 16.05 16.96 51463-2a-52032 9.94 17.91 18.97 51857-2a-51949 10.1214.16 12.21 51857-2a-51990 10.36 12.37 16.47 51857-2a-52032 8.57 15.1819.01 51446-2a-51536 9.45 10.43 14.76 46801-2a-47923 8.86 12.15 13.10GFP 1.65

Example 5: Activity of ZFN in CD34+ Cells Using a BTX ElectroporationDevice, Separate mRNAs and Single mRNAs

The activity of two pairs of ZFN were tested in mobilized human CD34+cells isolated from human peripheral blood and in CD34+ cells isolatedfrom bone marrow. Briefly, the CD34+ cells were isolated from healthydonors as follows. Leukapheresis collections were platelet depleted bylow speed centrifugation and supernatant removal. Following plateletdepletion, the cells were labelled with anti-CD34 magnetic micro-beads(Miltenyi Biotec, Germany) and positively selected using the MiltenyiCliniMACS Plus Cell Separator System. Following selection, the positivefraction (enriched CD34+ HSPC) were washed and resuspended in culturemedium (i.e., X-VIVO 10 medium supplemented with 2 mM L-glutamine, 100ng/mL each of FMS-like tyrosine kinase 3-ligand (Flt-3L), stem cellfactor (SCF), and thrombopoietin (TPO)) at 1×106 cells/mL, andtransferred into VueLife culture bags (Saint-Gobain, Gaithersburg, Md.)and incubated at 37° C./5% CO2. For purification of CD34+ cells frombone marrow, collections were depleted of red blood cells (RBC) byhydroxyethyl starch sedimentation. Following RBC depletion, the cellswere labelled with anti-CD34 magnetic micro-beads (Miltenyi Biotec,Germany) and positively selected using the Miltenyi CliniMACS Prodigy.Following selection, the positive fraction (enriched CD34+ HSPC) werewashed and resuspended in culture medium (i.e., X-VIVO 10 mediumsupplemented with 2 mM L-glutamine, 100 ng/mL each of FMS-like tyrosinekinase 3-ligand (Flt-3L), stem cell factor (SCF), and thrombopoietin(TPO)) at 1×106 cells/mL, and transferred into VueLife culture bags(Saint-Gobain, Gaithersburg, Md.) and incubated at 37° C./5% CO2.

The pairs used were 51446/51536 (pair A) and SBS51857/51949 (pair B).The ZFNs were tested as mRNA introduced into the cells. Fortransfection, a BTX device was used. Briefly, 200,000 cells per samplewere suspended in BTXpress Electroporation solution (BTX) and mixed withthe RNA. The mixture was then pulsed for 4 msec at 250 volts andsubjected to cold shock conditions (30C overnight) prior to letting thecells recover at 37° C. Analysis of ZFN activity was carried out two orthree days post-transfection. The ZFNs were tested as single mRNAspecies, where a 2a self-cleaving peptide sequence was used between thetwo ZFN coding sequences, and the data is presented in FIG. 2. Inaddition, the same conditions were used to test 1 pair of ZFN where theprovision of the mRNAs was as two separate species, where each mRNAencoded a single ZFN. Table 10a and Table 10b below shows the activityresults (% NHEJ or indels) for the single mRNA approach versus two mRNAs(data depicted is from several experiments).

TABLE 10a Comparison of single versus double mRNA species (% indels)mRNA used 2.0 μg 4.0 μg 51857_2a_51949, exp. #1 56.0 64.751857_2a_51949, exp. #2 61.2 73.9 51857 + 51949 83.1 80.5

TABLE 10b Comparison of single versus double mRNA species (% indels)mRNA used 0.5 μg 2.0 μg 4.0 μg 51857_2a_51949, exp. #3 32.0 51.5 58.751857 + 51949, exp #3 40.0 58.4 63.1

We also tested expression of human gamma globin and human beta globinusing TaqMan® according to standard protocols. The results were thennormalized as a ration of HBG (gamma) over HBB (beta), and are depictedin FIG. 3, again comparing two pairs of ZFN: 51446/51536 (pair A) andSBS51857/51949 (pair B). In addition, expression of HBG and HBB werealso measured comparing the provision of the ZFN pair as a single mRNA,where the sequences encoding each ZFN in the pair are separated by a 2aself-cleaving peptide sequence, with conditions where the mRNA encodingeach ZFN was supplied separately. The data demonstrated that under theseconditions, the B pair, SBS51857/51949, was more active in cleaving theBCL11a target and in causing an increase in HBG expression.

The ZFN pairs were tested in bone-marrow derived CD34+ cells and bothpairs were again found to be active (see FIG. 4a ). Activity for theSBS51857/51949 pair was also tested where the ZFNs were supplied on asingle mRNA with a 2a or as two separate mRNAs as described above (FIG.4b ). The SBS51857/51949 pair demonstrated higher activity than the51446/51536 pair.

Example 7: Specificity Analysis Unbiased Capture Analysis:

The capture assay is based on the observation that co-introduction of anuclease and a duplex DNA donor into a target cell results in the“capture” of donor into a fraction of the resultant genomic break sitesvia the NHEJ DNA repair pathway (Orlando et al. (2010) Nucleic AcidsRes. 38(15):e152; Gabriel et al. (2011) Nat Biotechnol. 29:816-23). Notethat this capture event is not homology driven (indeed the duplex DNAdonor does not contain any homology to the human genome). Note furtherthat the mRNA encoding the ZFNs cannot be captured into the DNA break,solely the duplex DNA donor can. Once trapped the duplex genomerepresents a permanent tag of the cleavage event. After isolation ofgenomic DNA, sites of capture may be identified via primer extensionfrom the donor into flanking genome sequence, followed by adapterligation, PCR, and sequencing of the resulting donor-genome junctions.

Briefly, the capture analysis studies were conducted in the K562 cellline to maximize donor delivery, ZFN expression, and donor capture intoDSB sites; the cells were electroporated with the ZFN-encoding mRNA andthe oligonucleotide duplex donor. Separately, BM and PB-derived CD34+cells were electroporated using the Maxcyte device as described abovewith the ZFN-encoding mRNA. The duplex donor oligonucleotide used isshown below (SEQ ID NOs: 37 and 38):

5′ NNNNAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCNNNNNAGTGTGGAAAATCTCTAGCAG 3′3′ TCATCACACACGGGCAGACAACACACTGAGACCATTGATCTCTAGGGAGTCTGGGAAAATCAGNNNNNTCACACCTTTTAGAGATCGTCNNNN 5′

The NNNN at the 5′ end of each strand indicates a random, single strandtetramer overhang, while the underlined NNNN indicates random duplexsequence that served as a bar code to differentiate between otherwiseidentical integration events. Triplicate samples were prepared for eachcombination of oligo and mRNA. On days 7 and 14 post-transfectiongenomic DNA was isolated (Qiagen DNeasy Blood and Tissue Kit), and 1 μg(330000 genomes/replicate) was used as input for the amplificationprotocol. Samples were then processed essentially as described(Paruzynski et al. (2010) Nat. Protocols. 5:1379-1395). Amplicons werepurified using a QIAquick PCR Purification Kit (Qiagen), and amplifiedby PCR to introduce barcodes and adapters for deep sequencing on theIllumina platform. Final products were quantified, pooled and sequencedon a MiSeq Instrument (Illumina) using a v2 300 cycle sequencing kitwith paired-end 150 bp reads and 8 bp dual index reads to detect thebarcodes on each end of the amplicon. This effort yielded a set ofcandidate off-target loci that were then genotyped in BM- or PB-derivedCD34 cells The results identified for pair A (SBS #51446/51536) areshown in FIG. 10a , while the results for pair B (SBS #51857/51949) areshown in FIG. 10b . The data is further summarized below in Table 11.

TABLE 11 Off target analysis for pair A and pair B, CD34+ from BoneMarrow A 250 ug/ml 63% 21 9% B 125 ug/ml 63% 9 3% B 250 ug/ml 74% 2017% 

Similar studies were performed on CD34+ cells derived from PB, and Table12 below summarized the results found.

TABLE 12 Off target analysis for pair A and pair B, CD34+ fromPeripheral Blood Cumulative ZFNs Dose On-Target # off-targets ActivitySt 4 120 ug/ml 60% 11 4% St 5 120 ug/ml 56% 10 3%

Example 6: Activity of ZFN in CD34+ Cells Using a MaxcyteElectroporation Device, Separate mRNAs and Single mRNAs

We next ran larger scale experiments using the ZFN pairs in CD34+ cellsusing a Maxcyte GT electroporation device. Mobilized CD34+ cellsisolated peripheral blood from normal donors as described above (PB)were tested as follows: cells (3 million per sample) were resuspended inRT Maxcyte EP buffer to a final concentration of 30 e6 cells/mL. Cellswere mixed with mRNA and electroporated using the program specified bymanufacturer. Cells were allowed to recover briefly at 37° C. for twentyminutes, then diluted and subjected to cold shock conditions (30° C.overnight) prior to letting the cells recover at 37° C. Activity wasanalyzed two to three days later. Experiments were done with both the Apair and the B pair as previously, where each pair was introduced on asingle mRNA (FIG. 5A). The ZFN pair B (SBS51857/51949) was also testedas a single mRNA or two separate mRNAs as described above (FIG. 5b ). Inthese experiments, the B pair showed the highest activity.

For bone marrow derived cells, successful editing required more mRNA,but high activity was observed (see FIG. 6). The cells were analyzed forHBG and HBB expression as described above and the HBG/HBB ratio comparedto the percent indel activity at day 0 (FIG. 7). There was a goodagreement between higher levels of HBG expression and higher indelactivity.

More analyses were done using the Maxcyte protocol (FIGS. 8 and 9) whereit was found that although the frequency of indels wasn't always thesame in each experiment, the relative activity (e.g., 2a SBS51857/51949construct higher than the separate mRNAs) was observed in each run.

Example 7: Engraftment of Edited Human CD34+ Cells in Mice

As described above, CD34+ human cells are treated with mRNAs encodingthe +58 enhancer specific ZFNs and then engrafted into NSG mice. CD34+cells are obtained from healthy human volunteers. In some cases, CD34+mobilization strategies were done, using either G-CSF (Neupogen®) orG-CSF+ Plerixafor (Mozobil®) prior to apheresis. The G-CSF isadministered daily for the four days prior to apheresis according tomanufacturer's instructions, and if Plerixafor was used, it wasadministered on the final evening prior to harvest, again according tomanufacturer's instructions. The apheresis is performed by standardmethods. CD34+ cells were enriched from the mobilized PBMC leukopaksusing a Miltenyi CliniMACs system by standard methods and according tomanufacturer's instructions.

Capped and poly-adenylated mRNAs encoding the ZFNs are synthesized usingAmbion mMessage mMachine® T7 ultra kit as instructed by the manufacturerand then electroporated into the CD34+ cells using either a Maxcyte GTsystem or a BTX ECM830 electroporator, both according to manufacturer'sinstructions.

NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ mice are used to receive theCD34+ transplant. One day (16-24 hours) prior to implantation, the miceare subject to sub lethal irradiation (300 RAD). The ZFN-treated CD34+cells from above are transplanted into the irradiated mice through atail vein injection, where 1 million cells in 0.5 mL PBS-0.1% BSA weregiven per mouse.

For this experiment, CD34+ cells arc electroporated with mRNAs encodingthe 46801/47923 pair. Genes encoding the ZFNs are cloned together in asingle open reading frame separated by a sequence encoding a 2Aself-cleaving peptide. GFP was used as a control. Followingtransplantation into the mice, samples are taken at either 4, 8 and 12weeks post-transplant to observe the level of human cell specificmarking in cells.

The ZFN-edited CD34+ cells engraft and differentiate, and levels ofengraftment are similar between the edited cells as compared to theunedited controls.

Example 8: Activity of ZFNs in Patient Derived Cells

The activity of the ZFN were tested in mobilized human CD34+ cellsisolated from human peripheral blood and in CD34+ cells isolated frombone marrow. HSPC from five β thalassemia subjects (designated P11, P18,P04, P08 and P19) were mobilized and purified as described (Yannaki etal. (2012) Mol Ther 20(1):230). In both experiments 200,000 CD34+ cellswere electroporated two days after thawing, using a BTX electroporator(Holliston, Mass., Voltage=250 V, pulse length=5 ms) in 100 μL of BTXExpress electroporation solution. Transfections used either 4 μg ofgreen fluorescent protein (GFP) encoding mRNA (as a control forelectroporation efficiency, and to control for nonspecific effects ofelectroporation itself, and for nonspecific effects of introducing mRNAinto the cells), or 4 μg and 8 μg of SB 51857-2a-51949 mRNA.

In a small scale transfection using the BTX electroporator these SB51857/51949 mRNA amounts resulted in equivalent target gene modificationand γ-globin activation to the 80-120 ug/ml concentrations used in thelarger scale MaxCyte device transfections.

Following electroporation, a transient overnight culture at 30° C. wasperformed. Cells were cultured for an additional 48 hours at 37° C.whereupon in vitro differentiation was initiated and cell aliquots wereharvested for analysis of DNA modification. After transfection, cellswere cultured in X Vivo 10 medium (Lonza, Walkersville, Md.)supplemented with the CC 100 cytokine cocktail (Stem Cell Technologies,Vancouver, Canada).

BCL11A gene modification was measured by MiSeq deep sequencing 72 hoursafter electroporation, at the time when the in vitro differentiation wasstarted (therefore d3 post-transfection was d0 of the differentiation)and at day 14 of the erythroid differentiation. The results are shown inTable 13.

The patient derived CD34+ cell samples we obtained had been frozen twiceprior to use, and therefore some of these sample exhibited reducedviability and cell growth upon thawing. Low viability post-thaw has beenobserved to coincide with higher reduction in viability aftertransfection and with lower target gene modification, especially at theearly time points, when the DNA from non-transfected dead cells is stillpresent. As an SB-ZFN transfection independent indicator of cellviability after transfection, Table 13 shows cell viability for eachcell source in the control sample which was transfected with GFP mRNA.The table shows that patient cell samples P18 in experiment 1 had muchlower viability (38%) than the other two samples (71% and 80%) in thisexperiment and did not reach the same modification levels of ˜70%alleles modified. Similarly in the second experiment at day 3 patientcell sample P08 showed very poor viability (22%) and very low earlymodification levels and suboptimal modification levels even afterexpansion and outgrowth of the healthy cells.

TABLE 13 BCL11A Gene Modification Analysis by MiSeq GFP Control TargetGene Cell Viability d 3 SB-mRENH Modification (%) source (%) mRNA (μg)Day 3 Day 14 Experiment WT 71 4 70 69 1 8 72 69 P11 80 4 70 71 8 73 73P18 38 4 45 53 8 51 59 Experiment WT 89 4 71 72 2 8 77 77 P04 62 4 48 628 53 67 P08 22 4 19 62 8 32 68 P19 68 4 ND 69 8 ND 73 ND = no data, WT =wild type

These data show that disruption of the BCL11A enhancer followingelectroporation of SB ZFN mRNA into G CSF mobilized, purified HSPCs fromhealthy donors and from thalassemia patients with β thalassemia occurredin most samples within the range expected for clinical samples. As aconsequence of the low viability of some of the samples, enhancerdisruption in P18 and P08 was lower than in cells from healthy donorvolunteers, and consequently the two samples with low viability wereomitted from the analyses below. Importantly, samples from subjects thatexhibited robust cell viability (e.g. samples P11 and P04) alsoexhibited gene modification levels equivalent to those seen in the wildtype cells.

Levels of α and γ globin mRNA isolated from erythroid progeny of CD34+HSPC from subjects with β thalassemia showed an increase in fetal (γ)globin levels following treatment with SB ZFN mRNA when analyzed by RTqPCR (FIG. 11). Fetal γ globin mRNA levels are shown normalized relativeto a globin mRNA since the thalassemia cells expressed low or no βglobin mRNA.

Erythroid progeny of CD34+ HSPCs from subjects with β thalassemiatreated with SB ZFN mRNA reveal the anticipated increase in the ratio offetal (γ) globin mRNA to α globin mRNA, reaching gamma-globin toalpha-globin ratios similar to those seen in the wild type donor cells,in particular in the patient samples that showed good viability afterthawing and consequently target gene modification levels comparable tothose in wild type cells.

Reverse phase HPLC was then used to determine whether modification ofthe BCL11A erythroid enhancer elevates fetal hemoglobin at the proteinlevel.

The gamma globin (sum of the Agamma and Ggamma peaks)/alpha globinratios for the two experiments showed a clear elevation of fetal globinprotein was observed in red blood cells (RBCs) derived from healthyvolunteers and thalassemia patients upon SB ZFN disruption of the BCL11Aenhancer, even though the untreated gamma/alpha ratios in thethalassemia cells especially in β0/β0 cells are usually well above thosein the wild-type (wt) cells. Analysis of fetal to adult globin ratios incells from patients with β thalassemia major is complicated by the factthat the tetramerization and precipitation of α globin in unmodifiedcells eliminates it from the HPLC analyzable pool.

Thus, solely α globin tetramerized with residual β globin (in patientswith β+ thalassemia) and γ globin, or solely α globin tetramerized withγ globin (in β0/β0 cells) can be revealed in the assay. Therefore, if γglobin protein levels increase, productively tetramerized a globinprotein levels can increase as well and the γ/α globin protein ratiounderestimated the increase in γ globin protein levels. Another ratiothat was useful to examine in wild type cells was the γ globin/β likeprotein ratio (the latter being the sum of the two γ globin proteinlevels plus δ globin plus β globin). However in thalassemia patientcells, particular in the β0/β0 cells, this ratio was usually over 90%even in non ZFN treated cells and even substantial increases in γ globinprotein levels after ZFN treatment did not markedly increase this ratio.

Example 9: Analysis of Modified Allele Distribution and Effect on FetalGlobin in ZFN Treated Wild Type CD34+ Cells

We also evaluated erythroid cells derived from CD34+ cells treated withSB ZFN, at the single cell level with respect to two endpoints: (1)distribution of alleles of BCL11A erythroid enhancer region rated by theaction of the ZFNs between individual cells, and (2) effect of thedistribution of individual alleles (wild type and genetically modified)on levels of fetal globin (as gauged by the γ/β globin mRNA ratio).Thus, single CD34+ cells found in SB ZFN HSPC were sorted anddifferentiated in vitro. The resulting single cell derived colonies ofhemoglobinized cells were harvested individually; genomic DNA (gDNA) foreach of the colonies was sequenced at the SB ZFN target region in theBCL11A erythroid enhancer to determine whether the locus has beendisrupted, and if yes, what precise allelic form of the locus wasgenerated. Further, total RNA was isolated from the same colony, andglobin expression levels in these individual clones were analyzed byreal time reverse transcription quantitative polymerase chain reaction(RT qPCR).

Methods

For single cell studies, transfected SB ZFN HSPC cells were thawed at37° C., added to 10 mL X VIVO at room temperature, and spun down at450×g for 5 minutes at room temperature. Cell pellets were resuspendedat 1·x 106/mL in X VIVO media supplemented with Flt3L, TPO, and SCF (100ng/mL each), penicillin (100 U/mL), and streptomycin (100 μg/mL). Afterovernight culture in a 24 well non tissue culture-treated plate at 37°C., 5% CO2 in a humidified incubator, cells were collected, spun down,and resuspended in phosphate buffered saline (PBS) supplemented with0.5% bovine serum albumin at 2×106/mL. Cells were sorted into Step 1erythroid culture media (200 μL/well) in 96 well U bottom non-TC treatedplates at 2 cells/well using FACS Aria III. Step 1 erythroid culturemedia consisted of Glutamax containing Iscove's Modified Dulbecco'sMedium (IMDM) supplemented with 100 U/mL penicillin, 100 μg/mLstreptomycin, 5% human AB+ plasma, 330 μg/mL human holo transferrin, 20μg/mL human insulin, 2 U/mL heparin, 3 U/mL recombinant humanerythropoietin, 100 ng/mL SCF, 5 ng/mL IL 3, and 1 μM/mL hydrocortisone.After 7 days of culture at 37° C., 5% C02 in Step 1 erythroid culturemedia, 150 μL of media per well was removed and replaced with 100 μLStep 2 erythroid culture media, which was similar to Step 1 media butwithout the addition of IL 3 and hydrocortisone. After 4 additional daysof culture, 100 μL media per well was removed and replaced with 100 μLStep 3 media, which was similar to Step 2 media but without SCF.

On Day 14 post differentiation, 10 μL of cell suspension per well washarvested for deep sequencing. Furthermore, 100 μL of media per well wasremoved and replaced with 100 μL of fresh Step 3 media. Remaining cellswere cultured for 3 more days when 50 μL of cell suspension per well wascollected, stained with an equal volume of NucRed (2 drop/mL in PBS 0.5%BSA), and enumerated on Guava easyCyte for cellularity and enucleationrate. Remaining cells were spun down, wash once with PBS, and lysed in20 μL high performance liquid chromatography-(HPLC) grade water. Celldebris was removed by centrifugation (10,000×g, 15 min, 4° C.).Hemolysate was stored at 70° C. until ready for globin chain analysis byreverse phase ultra performance liquid chromatography (UPLC).

Gene modification efficiency was assessed by deep DNA sequencing. Theregion of interest (containing the ZFN binding site within the BCL11Aerythroid enhancer region) is polymerase chain reaction-(PCR) amplifiedand the level of modification is determined by paired end deepsequencing on an Illumina MiSeq. To generate libraries compatible withthe Illumina MiSeq sequencing platform, adaptors, barcodes, and flowcell binder (short DNA sequence) were attached to the target specificamplicons using two sets of fusion primers in sequential polymerasechain reactions. The following primers are used for the MiSeq AdaptorPCR (the underlined portions are BCL11A erythroid enhancer specificsequences):

PRJIYLFN f: (SEQ ID NO: 35)5′ACACGACGCTCTTCCGATCTNNNNAGTCCTCTTCTACCCCACC and  PRJIYLFN r:(SEQ ID NO: 36) 5′GACGTGTGCTCTTCCGATCTCTACTCTTAGACATAACACACC.Individual single cell derived erythroid cultures were harvested, andgDNA was extracted using QuickExtract™ (Epicentre) for genotypinganalysis using deep sequencing on the Illumina platform.

For globin chain analysis by reverse phase UPLC, 5 μL of hemolysate wasinjected onto a Waters Acquity UPLC Protein BEH C4 Column (300 A, 1.7microm, 2.1 mm×100 mm). Elution was obtained at RT with a flow rate of0.2 mL/min using an 18 minute linear gradient of 38% to 42.5%acetonitrile in water with trifluoroacetic acid constant at 0.1%.Elution was followed at 220 nm. Area percentage for specific globinchains, γ, β or α, representing the amount of each specific globinchains, was quantitated using Agilent OpenLAB software.

Results

For this study, 120 or 80 μg/ml SB ZFN was transfected into CD34+ cellsto generate SB ZFN HSPC cells and cell samples were collected at threedays post transfection and analyzed for the levels of BCL11A erythroidenhancer region disruption by deep sequencing. This revealedapproximately 67% modified BCL11A alleles in the SB ZFN transfectedsample for experiment 1 and 58% for experiment 2 (Table 14).

To perform such a single cell analysis, individual cells in SB ZFN HSPCfrom two different experiments (1 and 2), were sorted and plated in 96well plates and underwent erythroid differentiation in vitro, andindividual single cell cultures were analyzed by high throughput DNAsequencing as well as globin expression analysis by UPLC, respectively.

The MiSeq genotyping results for the single cell cultures are summarizedin Table 14. For each lot of SB ZFN treated HSPC cells, between 200 300individual single cell erythroid cultures were analyzed. Of all theclones with clear phenotypes (mixed clones excluded, 205 clones forexperiment 1 and 265 clones for experiment 2), 28 or 36% are wild typeclones (+/+), 14 or 11% are heterozygous clones (+/), and 58 or 52% arehomozygous (/) clones, for experiment 1 and experiment 2, respectively.Of all alleles in the single cell erythroid cultures derived from SB ZFNtreated cells, 65% or 58%, for experiments 1 and 2, respectively, weredisrupted at the BCL11A erythroid enhancer locus. Of all the single cellerythroid cultures bearing any modified alleles, 81% (experiment 1) or82% (experiment 2) of the modified cell clones had both BCL11A allelesdisrupted.

TABLE 14 Genotyping of Single Cell Erythroid Cultures Derived from SBZFN Transfected HSPC Experiment number SB-ZFN-HSPC Lot #1 #2 Totalclones examined 205 265 (Total alleles) (410) (530) Wild-type Clones  57 96 (+/+) (28%) (36%) Heterozygous Clones  28  30 (Monoallelic Modified)(14%) (11%) (+/−) Homozygous Clones 120 139 (Biallellic Modified) (58%)(52%) (−/−) Net Modified 148 169 (Heterozygous [+/−] + (72%) (63%)Biallelic modified [−/−]) Fraction of Modified Cells 81% 82% which areBiallelic modified BCL11A erythroid enhancer modification 67% 58% inpool before single cell culture (% of total alleles) % of Total Alleles65% 58% Net BC11A erythroid enhancer modification from single cell data(% of total alleles)

The data show that a pool of SB ZFN HSPC bearing 58-67% targeted BCL11Aerythroid enhancer modification was made up of 28-36% wild type cells,11-14% cells bearing a monoallelic modification, and 52-58% cellsbearing biallelic modification of the target locus.

Of the clones with clear genotypes, 152 and 172 clones (experiments 1and 2, respectively) were successfully differentiated into erythroidcells in vitro, as indicated by enucleation rate measured by NucRedstain. These single cell erythroid cultures were then subjected toreverse phase UPLC analysis to measure globin chain expression level.Colonies differed in their degree of erythroid maturation; somevariation in the γ/β globin ratio was expected even within coloniesbearing the same BCL11A erythroid enhancer genotype. Furthermore, afraction of the disrupted alleles of the BCL11A erythroid enhancer mayretain partial or complete function, potentially as a result ofretaining a GATA 1 binding site (Vierstra et al. (2015) Nat Methods12(10):927-30; Canver et al. (2015) Nature 527(7577):192-7).

As can be seen in the data (FIG. 13), the results revealed a clearcorrelation between the genotype of a colony for the BCL11A erythroidenhancer locus and its γ/β globin ratio. Specifically, colonies bearinga biallelic (homozygous) modification of BCL11A had a mean normalizedγ/β ratio of 35% and 39% for experiments 1 and 2, respectively, colonieswith a monoallelic (heterozygous) modification had a mean normalized γ/βratio of 19% and 13% for experiments 1 and 2, respectively, and wildtype colonies 14% and 13% for experiments 1 and 2, respectively. In atwo tailed t test with Welch's correction, the P value of the“homozygous vs wild type” comparison are <0.0001 for both experiments 1and 2; the P value of the “heterozygous vs homozygous” comparison are<0.0001 for experiment 1 and 0.0025 for experiment 2; and the P value ofthe “heterozygous vs wild type” comparison are 0.02 and 0.0002 for bothexperiments 1 and 2, respectively. In the graph below (FIG. 13), the γ/βand γ/α globin ratio is plotted for all the colonies assayed, sorted bythe genotyping class of BCL11A erythroid enhancer alleles.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed:
 1. A pharmaceutical composition comprising genetically modified cells, the genetically modified cells comprising cells comprising at least one zinc finger nuclease, the zinc finger nuclease comprising a zinc finger protein comprising 4, 5 or 6 fingers designated F1 to F4, F1 to F5 or F1 to F6, each finger comprising a recognition helix region that recognizes a target subsite wherein the protein is selected from the group consisting of (i) a protein comprising the recognition helix regions as follows: F1: (SEQ ID NO: 7) STGNLTN; F2: (SEQ ID NO: 5) TSGSLTR; F3: (SEQ ID NO: 2) DQSNLRA; and F4: (SEQ ID NO: 6) AQCCLFH;

(ii) a protein comprising the recognition helix regions as follows: F1: (SEQ ID NO: 2) DQSNLRA; F2: (SEQ ID NO: 3) RPYTLRL; F3: (SEQ ID NO: 8) SRGALKT; F4: (SEQ ID NO: 5) TSGSLTR; F5: (SEQ ID NO: 2) DQSNLRA; and F6: (SEQ ID NO: 6) AQCCLFH;

(iii) a protein comprising the recognition helix regions as follows: F1: (SEQ ID NO: 2) DQSNLRA; F2: (SEQ ID NO: 9) RNFSLTM; F3: (SEQ ID NO: 10) SNGNLRN or (SEQ ID NO: 11) SSYNLAN; F4: (SEQ ID NO: 5) TSGSLTR; F5: (SEQ ID NO: 2) DQSNLRA; and F6: (SEQ ID NO: 6) AQCCLFH;

(iv) a protein comprising the recognition helix regions as follows: F1: (SEQ ID NO: 13) RSDHLTQ; F2: (SEQ ID NO: 14) QSGHLAR; F3: (SEQ ID NO: 15) QKGTLGE; F4: (SEQ ID NO: 16) RHRDLSR; and F5: (SEQ ID NO: 17) RRDNLHS;

(v) a protein comprising the recognition helix regions as follows: F1: (SEQ ID NO: 19) RNDHRTT; F2: (SEQ ID NO: 20) QKAHLIR; F3: (SEQ ID NO: 15) QKGTLGE; F4: (SEQ ID NO: 25) LKRTLKR; and F5: (SEQ ID NO: 17) RRDNLHS;

(vi) a protein comprising the recognition helix regions as follows: F1: (SEQ ID NO: 13) RSDHLTQ; F2: (SEQ ID NO: 22) QRAHLTR; F3: (SEQ ID NO: 15) QKGTLGE or (SEQ ID NO: 24) QSGTRNH; F4: (SEQ ID NO: 23) HRNTLVR; and F5: (SEQ ID NO: 17) RRDNLHS;

(vii) a protein comprising the recognition helix regions as follows: F1: (SEQ ID NO: 13) RSDHLTQ; F2: (SEQ ID NO: 20) QKAHLIR; F3: (SEQ ID NO: 15) QKGTLGE or  (SEQ ID NO: 24) QSGTRNH; F4: (SEQ ID NO: 21) RGRDLSR; and F5: (SEQ ID NO: 17) RRDNLHS;

(viii) a protein comprising the recognition helix regions as follows: F1: (SEQ ID NO: 13) RSDHLTQ; F2: (SEQ ID NO: 14) QSGHLAR; F3: (SEQ ID NO: 24) QSGTRNH; F4: (SEQ ID NO: 16) QSSDLSR; and F5: (SEQ ID NO: 17) RRDNLHS.


2. The pharmaceutical composition of claim 1, wherein the cells comprise one or more polynucleotides encoding the zinc finger nuclease.
 3. The pharmaceutical composition of claim 1, wherein the genetically modified cells are hematopoietic stem cells or erythroid precursor cells.
 4. The pharmaceutical composition of claim 3, wherein the cells are human cells.
 5. The pharmaceutical composition of claim 1, wherein the genetic modification is selected from the group consisting of insertions, deletions and combinations thereof.
 6. The pharmaceutical composition of claim 1, wherein the genetic modification is within the +58 region of the BCL11A enhancer sequence.
 7. The pharmaceutical composition of claim 1, further comprising genetically modified progeny cells produced from the genetically modified cells.
 8. The pharmaceutical composition of claim 7, wherein the genetically modified progeny cells comprise red blood cell (RBC) precursor cells or RBCs.
 9. The pharmaceutical composition of claim 7, wherein the genetically modified cells exhibit increased expression of gamma and/or beta globin as compared to cells without the genomic modification.
 10. The pharmaceutical composition of claim 1, wherein the zinc finger nuclease comprises a pair of zinc finger nucleases.
 11. The pharmaceutical composition of claim 1, wherein the pair of zinc finger nucleases comprise left and right zinc finger nucleases.
 12. The pharmaceutical composition of claim 11, wherein the left zinc finger nuclease comprises the zinc finger protein of (i), (ii) or (iii) and the right zinc finger nuclease comprises the zinc finger protein of (iv), (v), (vi), (vii) or (viii).
 13. The pharmaceutical composition of claim 1, wherein the genetic modification comprises an insertion.
 14. The pharmaceutical composition of claim 13, wherein an exogenous sequence is inserted into the genome of the cells.
 15. The pharmaceutical composition of claim 1, wherein the genetic modification comprises a deletion.
 16. A method of increasing globin production in a subject, the method comprising: administering the pharmaceutical composition of claim 7 to the subject.
 17. The method of claim 16, wherein the subject is a human.
 18. The method of claim 17, wherein the pharmaceutical composition is administered to bone marrow of the subject and wherein the cell engrafts, differentiates and matures in the subject.
 19. The method of claim 16, wherein the subject has a hemoglobinopathy.
 20. The method of claim 19, wherein the hemoglobinopathy is a beta-thalassemia or sickle cell disease.
 21. A kit comprising the pharmaceutical composition of claim
 7. 